Sorbent particles for sample treatment

ABSTRACT

Novel sorbents, devices, kits and methods useful for sample treatment are disclosed herein.

This application is a continuation of U.S. application Ser. No.16/591,302, which claims the benefit of U.S. Provisional ApplicationSer. No. 62/740,279, filed Oct. 2, 2018. The contents of each areincorporated herein by reference in their entireties.

FIELD

The present disclosure relates to sorbents, devices, kits and methodsthat may be used, for example, for the treatment of a biological sampleof interest. More particularly, the present disclosure relates tosorbents, devices, kits and methods that may be used for removal ofmatrix molecules, including phospholipids, from a biological sample ofinterest by Solid Phase Extraction (SPE).

BACKGROUND

Biological samples (e.g., biological fluids, animal tissue, planttissue, food, etc.) contain matrix components (i.e., components of asample other than the analyte of interest), many of which can interferewith sample analyses, including liquid chromatography-mass spectrometry(LC-MS) analysis. For example, biological samples, such as biologicalfluids, including plasma, whole blood, oral fluids, and urine, amongothers, animal tissue, plant tissue, and certain foods can containlipids, cells (proteins, peptides, amino acids, cell structures andorganelles) and salts which can interfere with LC-MS separations anddetection. The interference often manifests itself as shifting retentiontimes, convoluted detector signal (co-elution and noisy baseline),signal suppression (ion suppression) and MS adducts (i.e. Na+ or Cl−adducts). Lipids, including phospholipids, can also lead to column andinstrument fouling during LC-MS analysis. Removing such matrixcomponents can be advantageous to improve signal to noise ratios anddata reproducibility. Matrix removal devices can also be used to enrichanalytes of very low concentration or to add specificity to a givenanalytical protocol (i.e. extraction).

SUMMARY

In various aspects, the present disclosure pertains to methods of sampletreatment that comprise: adding a sample fluid comprising a sample thatcomprises at least one target analyte to a sorbent that comprises asilica component, a C4-C60 alkyl component, and an organic copolymercomponent comprising at least one hydrophobic organic monomer and atleast one hydrophilic organic monomer, thereby resulting in sorbent withbound target analyte; and adding an elution solution that comprises aprotic solvent and a polar aprotic solvent to the sorbent, therebydesorbing the target analyte from the sorbent and forming a solution ofthe target analyte in the elution fluid.

In various aspects, the present disclosure pertains to kits thatcomprise a sorbent that comprises a silica component, a C4-C60 alkylcomponent, and an organic copolymer component comprising a hydrophobicorganic monomer and a hydrophilic organic monomer and one or more kitcomponents selected from (a) an elution solution that comprises a proticsolvent and a polar aprotic solvent (b) a pretreatment solution, (c) oneor more washing solutions, (d) a collection plate or barrel, (e) a capmat, (f) calibration and reference standards, and (g) Identificationtagging for each component.

In various aspects, the present disclosure pertains to inorganic/organichybrid sorbent particles that comprise (a) a core region that comprisesan organic copolymer comprising at least one hydrophobic organic monomerand at least one hydrophilic organic monomer and (b) a surface regionthat comprises a silica component.

In various aspects, the present disclosure pertains to inorganic/organichybrid sorbent particles that comprise (a) a core region that comprisesa silica component and (b) a surface region that comprises an organiccopolymer comprising at least one hydrophobic organic monomer and atleast one hydrophilic organic monomer.

In various aspects, the present disclosure pertains to inorganic/organichybrid sorbent particles that comprise an organic copolymer thatcomprises at least one hydrophilic organic monomer, at least onehydrophobic organic monomer, and at least one alkenyl-functionalizedorganosilane monomer, wherein the particles are modified by the additionof a C4-C60 alkyl component.

Additional aspects and embodiments of the present disclosure are setforth in the following enumerated paragraphs:

Aspect A1. Inorganic/organic hybrid sorbent particles comprising (a) acore region that comprises a silica component and (b) a surface regionthat comprises an organic copolymer comprising at least one hydrophobicorganic monomer and at least one hydrophilic organic monomer.

Aspect A2. The inorganic/organic hybrid sorbent particles of aspect A1,herein the core region consists of silica or comprises a material havingthe formula SiO₂/(R² _(p)R⁴ _(g)SiO_(t))_(n) or SiO₂/[R⁶(R²_(r)SiO_(t))_(m)]_(n) wherein R² and R⁴ are independently C1-C18aliphatic or aromatic moieties, R⁶ is a substituted or unsubstitutedC1-C18 alkylene, alkenylene, alkynylene or arylene moiety bridging twoor more silicon atoms, p and q are 0, 1 or 2, provided that p+q=1 or 2,and that when p+q=1, t=1.5, and when p+q=2, t=1; r is 0 or 1, providedthat when r=0, t=1.5, and when r=1, t=1; m is an integer greater than orequal to 2, and n is a number from 0.03 to 1.

Aspect A3. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A2, further comprising a C4-C60 alkyl component.

Aspect A4. The inorganic/organic hybrid sorbent particles of any ofaspects A1-3, wherein the core region is formed by hydrolyticallycondensing one or more silane compounds.

Aspect A5. The inorganic/organic hybrid sorbent particles of aspect A4,wherein the one or more silane compounds that are hydrolyticallycondensed comprise (a) one or more silane compounds of the formulaSiZ₁Z₂Z₃Z₄, where Z₁, Z₂, Z₃ and Z₄ are independently selected from Cl,Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C4 alkyl, although at mostthree of Z₁, Z₂, Z₃ and Z₄ can be C1-C4 alkyl and/or (b) one or morecompounds of the formula Si Z₁Z₂Z₃—R—SiZ₄Z₅Z₆, where Z₁, Z₂ and Z₃ areindependently selected from Cl, Br, I, C1-C4 alkoxy, C1-C4 alkylamino,and C1-C4 alkyl, although at most two of Z₁, Z₂ and Z₃ can be C1-C4alkyl, and where Z₄, Z₅ and Z₆ are independently selected from Cl, Br,I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C4 alkyl, although at most twoof Z₄, Z₅ and Z₆ can be C1-C4 alkyl.

Aspect A6. The inorganic/organic hybrid sorbent particles of aspect A4,wherein the one or more alkoxysilane compounds are selected fromtetraalkoxysilanes, bis(trialkoxysilyl)alkanes, andalkyl-trialkoxysilane.

Aspect A7. The inorganic/organic hybrid sorbent particles of any ofaspects A4-A6, wherein the one or more silane compounds that arehydrolytically condensed further comprise a C4-C60-alkyl-functionalizedorganosilane compound.

Aspect A8. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A6, wherein a previously formed core region is reacted with aC4-C60-alkyl-functionalized organosilane compound.

Aspect A9. The inorganic/organic hybrid sorbent particles of any ofaspects A7-A8, wherein the C4-C60-alkyl-functionalized organosilanecompound is a compound of the formula RSiZ₁Z₂Z₃, where R is C4-C60 alkyland where Z₁, Z₂ and Z₃ are independently selected from Cl, Br, I, C1-C4alkoxy, C1-C4 alkylamino, and C1-C4 alkyl, although at most two of Z₁,Z₂ and Z₃ can be C1-C4 alkyl.

Aspect A10. The inorganic/organic hybrid sorbent particles of any ofaspects A7-A8, wherein the C4-C60-alkylorganosilane is selected fromoctadecyltrimethoxysilane, octadecyltriethoxysilane,octadecyltrichlorosilane, octadecyltri(dimethylamino)silane,methyloctadecyldimethoxysilane, methyloctadecyldiethoxysilane,methyloctadecyldichlorosilane, methyloctadecyldi(dimethylamino)silane,dimethyloctadecylmethoxysilane, dimethyloctadecylethoxysilane,dimethyloctadecylchlorosilane, dimethyloctadecyldimethylaminosilane,diisopropyloctadecylmethoxysilane, diisopropyloctadecylethoxysilane,diisopropyloctadecylchlorosilane,diisopropyloctadecyldimethylaminosilane, octyltrimethoxysilane,octyltriethoxysilane, octyltrichlorosilane,octyltri(dimethylamino)silane, methyloctyldimethoxysilane,methyloctyldiethoxysilane, methyloctyldichlorosilane,methyloctyldi(dimethylamino)silane, dimethyloctylmethoxysilane,dimethyloctylethoxysilane, dimethyloctylchlorosilane,dimethyloctyldimethylaminosilane, diisopropyloctylmethoxysilane,diisopropyloctylethoxysilane, diisopropyloctylchlorosilane,diisopropyloctyldimethylaminosilane, butyltrimethoxysilane,butyltriethoxysilane, butyltrichlorosilane,butyltri(dimethylamino)silane, methylbutyldimethoxysilane,methylbutyldiethoxysilane, methylbutyldichlorosilane,methylbutyldi(dimethylamino)silane, dimethylbutylmethoxysilane,dimethylbutylethoxysilane, dimethylbutylchlorosilane,dimethylbutyldimethylaminosilane, diisopropylbutylmethoxysilane,diisopropylbutylethoxysilane, diisopropylbutylchlorosilane, anddiisopropylbutyldimethylaminosilane.

Aspect A11. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A10, wherein the core region further comprises analkenyl-functionalized organosilane species incorporated into the core.

Aspect A12. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A10, wherein an alkenyl-functionalized organosilane monomeris attached at the surface of the core region,

Aspect A13. The inorganic/organic hybrid sorbent particles of any ofaspects A3-A10, wherein the silane compounds that are hydrolyticallycondensed further comprise an alkenyl-functionalized organosilanespecies.

Aspect A14. The inorganic/organic hybrid sorbent particles of any ofaspects A11-A13, wherein the alkenyl-functionalized organosilane monomeris selected from 3-(trimethoxysilyl)propyl methacrylate (MAPTMOS), andvinyltriethoxysilane (VTES).

Aspect A15. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A14, wherein the surface region that comprises the organiccopolymer is formed by a process that comprises an additionpolymerization of the at least one hydrophobic organic monomer and theat least one hydrophilic organic monomer.

Aspect A16. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A14, wherein the surface region that comprises the organiccopolymer is formed by a process that comprises free radicalpolymerization of the at least one hydrophobic organic monomer and theat least one hydrophilic organic monomer.

Aspect A17. The inorganic/organic hybrid sorbent particles of aspectA16, wherein the living radical polymerization is selected fromReversible Addition-Fragmentation Chain Transfer Polymerization (RAFT)and Atom Transfer Radical Polymerization (ATRP).

Aspect A18. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A17, wherein the surface region that comprises the organiccopolymer is formed by a process that comprises forming the organiccopolymer and grafting the organic copolymer onto a surface of the coreregion (i.e., “grafting to”) or polymerizing the organic copolymer frominitiator sites on the surface of the core region (“grafting from”).

Aspect A19. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A18, wherein the hydrophilic organic monomer comprises amonomer having the following formula,

where n ranges from 1-3.

Aspect A20. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A19, wherein the hydrophobic organic monomer comprisesdivinylbenzene and, optionally, styrene.

Aspect A21. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises anorganic monomer that comprises one or more sulfonate groups.

Aspect A22. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more sulfonate groups.

Aspect A23. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises anorganic monomer that comprises one or more quaternary ammonium groups.

Aspect A24. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises anorganic monomer that comprises one or more —R₁N⁺R₂R₃R₄ groups, where R₁is a C1-C8 alkylene group, and R₂, R₃ and R₄ are C1-C8 alkyl groups andmay be the same or different.

Aspect A25. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more —R₁N⁺R₂R₃R₄ groups,where R₁ is a C1-C8 alkylene group, and R₂, R₃ and R₄ are C1-C8 alkylgroups and may be the same or different.

Aspect A26. The inorganic/organic hybrid sorbent particles of any ofaspects A24-A25, wherein w=1, x=0, y=0 and z=3.

Aspect A27. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises anorganic monomer that comprises one or more carboxyl groups.

Aspect A28. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more carboxyl groups.

Aspect A29. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises anorganic monomer that comprises one or more primary, secondary ortertiary amine groups.

Aspect 30. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more piperazine groups.

Aspect A31. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A30, wherein the organic copolymer further comprises one ormore C4-C60 alkyl groups.

Aspect A32. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A30, wherein the organic copolymer further includes aC4-C-60-alkyl functionalized unsaturated monomer.

Aspect A33. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A30, wherein a C4-C-60-alkyl-group is attached to the organiccopolymer.

Aspect A34. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A20, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more piperazine groups.

Aspect A35. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A30, wherein the organic copolymer further comprises one ormore C4-C60 alkyl groups.

Aspect A36. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A30, wherein the organic copolymer further includes aC4-C-60-alkyl functionalized unsaturated monomer.

Aspect A37. The inorganic/organic hybrid sorbent particles of any ofaspects A1-A30, wherein a C4-C-60-alkyl-group is attached to the organiccopolymer.

Aspect B1. Inorganic/organic hybrid sorbent particles comprising (a) acore region that comprises an organic copolymer comprising at least onehydrophobic organic monomer and at least one hydrophilic organic monomerand (b) a surface region that comprises a silica component.

Aspect B2. The inorganic/organic hybrid sorbent particles of aspect B1,wherein the surface region further comprises a C4-C60 alkyl component.

The inorganic/organic hybrid sorbent particles of aspect B1, wherein theat least one

hydrophilic organic monomer comprises a monomer having the followingformula, where n ranges from 1-3

Aspect B4. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B3, wherein the at least one hydrophobic organic monomercomprises divinylbenzene and, optionally, styrene.

Aspect B5. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises anorganic monomer that comprises one or more sulfonate groups.

Aspect B6. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more sulfonate groups.

Aspect B7. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises anorganic monomer that comprises one or more quaternary ammonium groups.

Aspect B8. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises anorganic monomer that comprises one or more —R₁—N⁺R₂R₃R₄ groups, where R₁is a C1-C8 alkylene group, and R₂, R₃ and R₄ are C1-C8 alkyl groups andmay be the same or different.

Aspect B9. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more —R₁—N⁺R₂R₃R₄ groups,where R₁ is a C1-C8 alkylene group, and R₂, R₃ and R₄ are C1-C8 alkylgroups and may be the same or different.

Aspect B10. The inorganic/organic hybrid sorbent particles of any ofaspects B8-B9, wherein R₁ is C1 alkyl, R₂ is C1 alkyl, R₃ is C1 alkyl,and R₄ is C4 alkyl.

Aspect B11. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises anorganic monomer that comprises one or more carboxyl groups.

Aspect B12. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more carboxyl groups.

Aspect B13. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises anorganic monomer that comprises one or more primary, secondary ortertiary amine groups.

Aspect B14. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B4, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more piperazine groups.

Aspect B15. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B14, wherein the surface region is formed by hydrolyticallycondensing one or more silane compounds at the surface region of thecore region.

Aspect B16. The inorganic/organic hybrid sorbent particles of aspectB15, wherein the one or more silane compounds that are hydrolyticallycondensed comprise (a) one or more silane compounds of the formulaSiZ₁Z₂Z₃Z₄, where Z₁, Z₂, Z₃ and Z₄ are independently selected from Cl,Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C4 alkyl, although at mostthree of Z₁, Z₂, Z₃ and Z₄ can be C1-C4 alkyl and/or (b) one or morecompounds of the formula Si Z₁Z₂Z₃—R—SiZ₄Z₅Z₆, where Z₁, Z₂ and Z₃ areindependently selected from Cl, Br, I, C1-C4 alkoxy, C1-C4 alkylamino,and C1-C4 alkyl, although at most two of Z₁, Z₂ and Z₃ can be C1-C4alkyl, and where Z₄, Z₅ and Z₆ are independently selected from Cl, Br,I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C4 alkyl, although at most twoof Z₄, Z₅ and Z₆ can be C1-C4 alkyl.

Aspect B17. The inorganic/organic hybrid sorbent particles of aspectB15, wherein the one or more silane compounds that are hydrolyticallycondensed are selected from tetraalkoxysilanes,bis(trialkoxysilyl)alkanes and alkyl-trialkoxysilanes.

Aspect B18. The inorganic/organic hybrid sorbent particles of aspectB15, wherein the one or more silane compounds that are hydrolyticallycondensed are selected from tetramethoxysilane, tetraethoxysilane,bis(triethoxysilyl)ethane and methyl-triethoxysilane.

Aspect B19. The inorganic/organic hybrid sorbent particles of any ofaspects B15-B18, wherein the one or more silane compounds that arehydrolytically condensed further comprise a C4-C60-alkyl-functionalizedorganosilane compound.

Aspect B20. The inorganic/organic hybrid sorbent particles of any ofaspects B15-B18, wherein the surface region is formed by hydrolyticallycondensing one or more silane compounds at the surface region of thecore region, followed by reaction with a C4-C60-alkyl-functionalizedorganosilane compound.

Aspect B21. The inorganic/organic hybrid sorbent particles of any ofaspects B19-B20, wherein the C4-C60-alkyl-functionalized organosilanecompound is a compound of the formula RSiZ₁Z₂Z₃, where R is C4-C60 alkyland where Z₁, Z₂ and Z₃ are independently selected from Cl, Br, I, C1-C4alkoxy, C1-C4 alkylamino, and C1-C4 alkyl, although at most two of Z₁,Z₂ and Z₃ can be C1-C4 alkyl.

Aspect B22. The inorganic/organic hybrid sorbent particles of any ofaspects B19-B20, wherein the C4-C60-alkylorganosilane is selected fromoctadecyltrimethoxysilane, octadecyltriethoxysilane,octadecyltrichlorosilane, octadecyltri(dimethylamino)silane,methyloctadecyldimethoxysilane, methyloctadecyldiethoxysilane,methyloctadecyldichlorosilane, methyloctadecyldi(dimethylamino)silane,dimethyloctadecylmethoxysilane, dimethyloctadecylethoxysilane,dimethyloctadecylchlorosilane, dimethyloctadecyldimethylaminosilane,diisopropyloctadecylmethoxysilane, diisopropyloctadecylethoxysilane,diisopropyloctadecylchlorosilane,diisopropyloctadecyldimethylaminosilane, octyltrimethoxysilane,octyltriethoxysilane, octyltrichlorosilane,octyltri(dimethylamino)silane, methyloctyldimethoxysilane,methyloctyldiethoxysilane, methyloctyldichlorosilane,methyloctyldi(dimethylamino)silane, dimethyloctylmethoxysilane,dimethyloctylethoxysilane, dimethyloctylchlorosilane,dimethyloctyldimethylaminosilane, diisopropyloctylmethoxysilane,diisopropyloctylethoxysilane, diisopropyloctylchlorosilane,diisopropyloctyldimethylaminosilane, butyltrimethoxysilane,butyltriethoxysilane, butyltrichlorosilane,butyltri(dimethylamino)silane, methylbutyldimethoxysilane,methylbutyldiethoxysilane, methylbutyldichlorosilane,methylbutyldi(dimethylamino)silane, dimethylbutylmethoxysilane,dimethylbutylethoxysilane, dimethylbutylchlorosilane,dimethylbutyldimethylaminosilane, diisopropylbutylmethoxysilane,diisopropylbutylethoxysilane, diisopropylbutylchlorosilane, anddiisopropylbutyldimethylaminosilane.

Aspect B23. The inorganic/organic hybrid sorbent particles of any ofaspects B19-B20, wherein the one or more silane compounds that arehydrolytically condensed comprise a mixture of octadecyltriethoxysilane,tetraethoxysilane and bis(triethoxysilyl)ethane at a surface region ofthe core region.

Aspect B24. The inorganic/organic hybrid sorbent particles of any ofaspects B1-B23, further comprising an alkenyl-functionalizedorganosilane monomer.

Aspect B25. The inorganic/organic hybrid sorbent particles of aspectB24, wherein the organic copolymer further comprises thealkenyl-functionalized organosilane monomer.

Aspect B26. The inorganic/organic hybrid sorbent particles of aspectB24, wherein the alkenyl-functionalized organosilane monomer is attachedat the surface of the core region,

Aspect B27. The inorganic/organic hybrid sorbent particles of any ofaspects B24-B26, wherein the alkenyl-functionalized organosilane monomeris selected from 3-(trimethoxysilyl)propyl methacrylate (MAPTMOS), andvinyltriethoxysilane (VTES).

Aspect C1. Inorganic/organic hybrid sorbent particles comprising anorganic copolymer that comprises at least one hydrophilic organicmonomer, at least one hydrophobic organic monomer, and at least onealkenyl-functionalized organosilane monomer, wherein the particles aremodified by the addition of a C4-C60 alkyl component.

Aspect C2. The inorganic/organic hybrid sorbent particles of aspect C1,wherein the particles are modified by hydrolytic condensation of aC4-C60-alkyl-functionalized organosilane compound.

Aspect C3. The inorganic/organic hybrid sorbent particles of aspect C2,wherein the C4-C60-alkyl-functionalized organosilane compound is acompound of the formula RSiZ₁Z₂Z₃, where R is C4-C60 alkyl and where Z₁,Z₂ and Z₃ are independently selected from Cl, Br, I, C1-C4 alkoxy, C1-C4alkylamino, and C1-C4 alkyl, although at most two of Z₁, Z₂ and Z₃ canbe C1-C4 alkyl.

Aspect C4. The inorganic/organic hybrid sorbent particles of aspect C2,wherein the C4-C60-alkylorganosilane is selected fromoctadecyltrimethoxysilane, octadecyltriethoxysilane,octadecyltrichlorosilane, octadecyltri(dimethylamino)silane,methyloctadecyldimethoxysilane, methyloctadecyldiethoxysilane,methyloctadecyldichlorosilane, methyloctadecyldi(dimethylamino)silane,dimethyloctadecylmethoxysilane, dimethyloctadecylethoxysilane,dimethyloctadecylchlorosilane, dimethyloctadecyldimethylaminosilane,diisopropyloctadecylmethoxysilane, diisopropyloctadecylethoxysilane,diisopropyloctadecylchlorosilane,diisopropyloctadecyldimethylaminosilane, octyltrimethoxysilane,octyltriethoxysilane, octyltrichlorosilane,octyltri(dimethylamino)silane, methyloctyldimethoxysilane,methyloctyldiethoxysilane, methyloctyldichlorosilane,methyloctyldi(dimethylamino)silane, dimethyloctylmethoxysilane,dimethyloctylethoxysilane, dimethyloctylchlorosilane,dimethyloctyldimethylaminosilane, diisopropyloctylmethoxysilane,diisopropyloctylethoxysilane, diisopropyloctylchlorosilane,diisopropyloctyldimethylaminosilane, butyltrimethoxysilane,butyltriethoxysilane, butyltrichlorosilane,butyltri(dimethylamino)silane, methylbutyldimethoxysilane,methylbutyldiethoxysilane, methylbutyldichlorosilane,methylbutyldi(dimethylamino)silane, dimethylbutylmethoxysilane,dimethylbutylethoxysilane, dimethylbutylchlorosilane,dimethylbutyldimethylaminosilane, diisopropylbutylmethoxysilane,diisopropylbutylethoxysilane, diisopropylbutylchlorosilane, anddiisopropylbutyldimethylaminosilane.

Aspect C5. The inorganic/organic hybrid sorbent particles of aspect C1,wherein the alkenyl-functionalized organosilane monomer is selected from3-(trimethoxysilyl)propyl methacrylate (MAPTMOS) andvinyltriethoxysilane (VTES).

Aspect C6. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C5, wherein the hydrophilic organic monomer comprises amonomer having the following formula,

where n ranges from 1-3.

Aspect C7. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C6, wherein the hydrophobic organic monomer comprisesdivinylbenzene and, optionally, styrene.

Aspect C8. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises anorganic monomer that comprises one or more sulfonate groups.

Aspect C9. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more sulfonate groups.

Aspect C10. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises anorganic monomer that comprises one or more quaternary ammonium groups.

Aspect C11. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises anorganic monomer that comprises one or more —R₁N⁺R₂R₃R₄ groups, where R₁is a C1-C8 alkylene group, and R₂, R₃ and R₄ are C1-C8 alkyl groups andmay be the same or different.

Aspect C12. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more-R₁N⁺R₂R₃R₄ groups,where R₁ is a C1-C8 alkylene group, and R₂, R₃ and R₄ are C1-C8 alkylgroups and may be the same or different.

Aspect C13. The inorganic/organic hybrid sorbent particles of any ofaspects C11-C12, wherein w=1, x=0, y=0 and z=3.

Aspect C14. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises anorganic monomer that comprises one or more carboxyl groups.

Aspect C15. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more carboxyl groups.

Aspect C16. The inorganic/organic hybrid sorbent particles of any ofaspects C1-C7, wherein the organic copolymer further comprises anorganic monomer that comprises one or more primary, secondary ortertiary amine groups.

Aspect C17. The inorganic/organic hybrid sorbent particles of any ofaspects C1C-7, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more piperazine groups.

Aspect D1. A method of sample treatment, comprising: (a) adding a samplefluid comprising a sample that comprises at least one target analyte toa sorbent that comprises a silica component, a C4-C60 alkyl component,and an organic copolymer component comprising at least one hydrophobicorganic monomer and at least one hydrophilic organic monomer, therebyresulting in sorbent with bound target analyte; and (b) adding anelution solution that comprises a protic solvent and a polar aproticsolvent to the sorbent, thereby desorbing the target analyte from thesorbent and forming a solution of the target analyte in the elutionfluid.

Aspect D2. The method of aspect D1, wherein the sample further comprisesphospholipids, wherein adding the sample fluid to the sorbent results insorbent with bound phospholipids as well as the bound target analyte,and wherein adding the elution solution to the sorbent desorbs thetarget analyte from the sorbent while retaining at least 50% of thephospholipids on the sorbent.

Aspect D3. The method of any of aspects D1-D2, wherein the elutionsolution comprises a polar aprotic solvent selected from acetonitrile,acetone, tetrahydrofuran, methylene chloride, ethyl acetate,N,N-dimethylformamide, dimethyl sulfoxide and dimethyl ether.

Aspect D4. The method of any of aspects D1-D3, wherein the elutionsolution comprises a protic solvent selected from methanol, ethanol,1-propanol, 2-propanol, t-butanol, acetic acid, formic acid, aqueousammonia, methanolic ammonia, water, and blends thereof.

Aspect D5. The method of any of aspects D1-D2, wherein the elutionsolution comprises acetonitrile as a polar aprotic solvent and methanolas a protic solvent.

Aspect D6. The method of any of aspects D1-D5, wherein the elutionsolution comprises from 60 vol % to 100 vol % polar aprotic solvent andfrom 0 vol % to 40 vol % protic solvent.

Aspect D7. The method of any of aspects D1-D6, wherein the alkylcomponent of the sorbent is bound to the silica component.

Aspect D8. The method of any of aspects D1-D7, wherein the hydrophilicorganic monomer comprises a monomer having the following formula,

where n ranges from 1-3.

Aspect D9. The method of any of aspects D1-D8, wherein the hydrophobicorganic monomer comprises divinylbenzene and, optionally, styrene.

Aspect D10. The method of any of aspects D1-D9, wherein the sorbent isnot an ion exchange sorbent.

Aspect D11. The method of aspect D10, wherein the elution solutioncomprises acetonitrile and methanol in a ratio ranging from 70:30 to95:5 vol/vol.

Aspect D12. The method of any of aspects D10-D11, wherein the samplefluid is prepared by combining the sample with a pretreatment solutionthat comprises a pretreatment compound, water, and an organic solvent.

Aspect D13. The method of aspect D12, wherein the organic solvent is aprotic organic solvent.

Aspect D14. The method of any of aspects D12-D13, wherein thepretreatment compound is H₃PO₄, formic acid, acetic acid, sulfuric acid,or a blend thereof.

Aspect D15. The method of aspect D12, wherein the pretreatment solutioncomprises H₃PO₄, water and methanol.

Aspect D16. The method of aspect D12, wherein the pretreatment solutioncontains 3-5 wt % concentrated H₃PO₄ in water and methanol, which areprovided in a ratio ranging from 92:8 to 97:3 vol/vol and the sample iscombined with the pretreatment solution in a ratio ranging from 1:3 to1:5 vol/vol.

Aspect D17. The method of any of aspects D10-D11, wherein the samplefluid is prepared by combining the sample with a pretreatment solutionthat comprises a pretreatment compound and water.

Aspect D18. The method of aspect D17, wherein the pretreatment solutioncomprises H₃PO₄ and water.

Aspect D19. The method of aspect D17, wherein the pretreatment solutioncontains 3-5 wt % concentrated H₃PO₄ in water and the sample is combinedwith the pretreatment solution in a ratio ranging from 2:3 to 2:1vol/vol.

Aspect D20. The method of any of aspects D17-D19, further comprisingadding a washing solution to the sorbent after adding the sample fluidand before adding the elution solution, the washing solution comprisingwater and an organic solvent.

Aspect D21. The method of aspect D20, wherein the washing solutioncomprises water and a protic organic solvent.

Aspect D22. The method of aspect D20, wherein the washing solutioncomprises water and methanol.

Aspect D23. The method of aspect D20, wherein the washing solutioncomprises water and methanol in a ratio ranging from 90:10 to 98:2.

Aspect D24. The method of any of aspects D1-D9, wherein the sorbent is astrong cation exchange sorbent.

Aspect D25. The method of aspect D24, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore sulfonate groups.

Aspect D26. The method of aspect D24, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more sulfonate groups.

Aspect D27. The method of any of aspects D24-D26, wherein the targetanalyte is a basic target analyte.

Aspect D28. The method of any of aspects D1-9, wherein the sorbent is aweak anion exchange sorbent.

Aspect D29. The method of aspect D28, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore primary, secondary or tertiary amine groups.

Aspect D30. The method of aspect D28, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more piperazine groups.

Aspect D31. The method of any of aspects D28-D30, the target analyte isa strong acid.

Aspect D32. The method of any of aspects D24-D31, wherein the elutionsolution comprises water and a basic compound.

Aspect D33. The method of aspect D32, wherein the basic compoundselected from ammonium hydroxide (NH₄OH), NaOH, KOH, ammoniumbicarbonate and combinations thereof.

Aspect D34. The method of any of aspects D24-D33, wherein the elutionsolution comprises acetonitrile and methanol in a ratio ranging from60:40 to 100:0 vol/vol.

Aspect D35. The method of any of aspects D24-D34, wherein the elutionsolution comprises NH₄OH (aq), acetonitrile and methanol.

Aspect D36. The method of any of aspects D24-D35, wherein elutionsolution comprises 3-8 wt % concentrated NH₄OH in water.

Aspect D37. The method of any of aspects D24-D36, wherein the samplefluid comprises the sample combined with a pretreatment solutioncomprising an organic solvent and one or more pretreatment compounds.

Aspect D38. The method of aspect D37, wherein the organic solvent is aprotic organic solvent.

Aspect D39. The method of aspect D37, wherein the protic organic solventis methanol.

Aspect D40. The method of any of aspects D37-D39, wherein the one ormore pretreatment compounds a selected from formic acid, carbonic acid,acetic acid.

Aspect D41. The method of aspect D40, wherein the one or morepretreatment compounds further comprise H₃PO₄.

Aspect D42. The method of aspect D37, wherein the pretreatment solutioncomprises methanol, formic acid and H₃PO₄.

Aspect D43. The method of aspect D37, wherein the pretreatment solutioncontains 1-4 wt % concentrated H₃P04 in water and 1-4 wt % formic acidin methanol and the sample is combined with the pretreatment solution ina ratio ranging from 1:3 to 1:6 vol/vol.

Aspect D44. The method of any of aspects D24-D36, wherein the samplefluid comprises the sample combined with a pretreatment solution thatcomprises water and a pretreatment compound.

Aspect D45. The method of aspect D44, wherein the pretreatment compoundis selected from H₃PO₄, formic acid, acetic acid, sulfuric acid, or ablend thereof.

Aspect D46. The method of aspect D44, wherein the pretreatment compoundis H₃PO₄.

Aspect D47. The method of aspect D44, wherein the pretreatment solutioncontains 2-8 wt % concentrated H₃PO₄ in water and the sample is combinedwith the pretreatment solution in a ratio ranging from 2:3 to 2:1vol/vol.

Aspect D48. The method of any of aspects D44-D47, wherein the methodfurther comprises adding a washing solution to the sorbent after addingthe sample fluid and before adding the elution solution, the washingsolution comprising an acidic compound and an organic solvent.

Aspect D49. The method of aspect D48, wherein the organic solvent is aprotic organic solvent.

Aspect D50. The method of aspect D48, wherein the organic solvent ismethanol.

Aspect D51. The method any of aspects D48-D50, wherein the acidiccompound is selected from formic acid, carbonic acid, and acetic acid.

Aspect D52. The method of aspect D48, wherein the washing solutioncomprises formic acid and methanol.

Aspect D53. The method of aspect D48, wherein the washing solutioncontains 1-4 wt % formic acid in methanol.

Aspect D54. The method of any of aspects D44-D47, wherein the methodfurther comprises (a) adding a first washing solution to the sorbentafter adding the sample fluid and before adding the elution solution,the first washing solution comprising an acidic compound and water and(b) adding a second washing solution to the sorbent after adding thefirst washing fluid and before adding the elution solution, the secondwashing solution comprising an organic solvent.

Aspect D55. The method of aspect D54, wherein the acidic compound isselected from formic acid, carbonic acid, and acetic acid.

Aspect D56. The method of aspect D54, wherein the acidic compound isformic acid.

Aspect D57. The method of any of aspects D54-D56, wherein the organicsolvent is a protic organic solvent.

Aspect D58. The method of any of aspects D54-D56, wherein the organicsolvent is methanol.

Aspect D59. The method of aspect D54, wherein the first washing solutioncomprises formic acid in water and the second washing solution comprisesmethanol.

Aspect D60. The method of aspect D54, wherein the first washing solutioncontains 1-4 wt % formic acid in water and the second washing solutioncomprises methanol.

Aspect D61. The method of any of aspects D1-D9, wherein the sorbent is astrong anion exchange sorbent.

Aspect D62. The method of aspect D61, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore quaternary ammonium groups.

Aspect D63. The method of aspect D61, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore —R₁N⁺R₂R₃R₄ groups, where R₁ is a C1-C8 alkylene group, and R₂, R₃and R₄ are C1-C8 alkyl groups and may be the same or different.

Aspect D64. The method of aspect D61, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more —R₁N⁺R₂R₃R₄ groups, where R₁ is a C1-C8 alkylene group, and R₂,R₃ and R₄ are C1-C8 alkyl groups and may be the same or different.

Aspect D65. The method of any of aspects D63-D64, wherein w=1, x=0, y=0and z=3.

Aspect D66. The method of any of aspects D61-D65, wherein the targetanalyte is an acidic target analyte.

Aspect D67. The method of any of aspects D1-D9, wherein the sorbent is aweak cation exchange sorbent.

Aspect 68. The method of aspect D67, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore carboxyl groups.

Aspect D69. The method of aspect D67, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more carboxyl groups.

Aspect D70. The method of any of aspects D67-D69, wherein the targetanalyte is a strong base or a quaternary amine compound.

Aspect D71. The method of any of aspects D61-D70, wherein the elutionsolution comprises water and an acidic compound.

Aspect D72. The method of aspect D71, wherein the acidic compound isselected from formic acid, acetic acid, and carbonic acid.

Aspect D73. The method of any of aspects D61-D72, wherein the elutionsolution comprises acetonitrile and methanol in a ratio ranging from65:35 to 85:15 vol/vol.

Aspect D74. The method of any of aspects D61-D73, wherein the elutionsolution comprises formic acid, acetonitrile and methanol.

Aspect D75. The method of any of aspects D61-D74, wherein elutionsolution comprises 1-4 wt % formic acid.

Aspect D76. The method of any of aspects D61-D75, wherein the samplefluid comprises the sample combined with a pretreatment solution thatcomprises an organic solvent and a pretreatment compound.

Aspect D77. The method of aspect D76, wherein the pretreatment compoundis selected from NH₄OH, ammonium bicarbonate, pyridine, piperazine, andcombinations thereof.

Aspect D78. The method of any of aspects D76-D77, wherein the organicsolvent is a protic organic solvent.

Aspect D79. The method of any of aspects D76-D77, wherein the organicsolvent is methanol.

Aspect D80. The method of aspect D76, wherein the pretreatment solutioncomprises NH₄OH in methanol.

Aspect D81. The method of aspect D76, wherein the pretreatment solutioncontains 3-8 wt % concentrated NH₄OH (aq) in methanol and the sample iscombined with the pretreatment solution in a ratio ranging from 1:2 to1:8 vol/vol.

Aspect D82. The method of any of aspects D61-D75, wherein the samplefluid comprises the sample combined with a pretreatment solution thatcomprises water and a pretreatment compound.

Aspect D83. The method of aspect D82, wherein the pretreatment compoundis selected from NH₄OH, ammonium bicarbonate, pyridine, piperazine, andcombinations thereof.

Aspect D84. The method of aspect D82, wherein the pretreatment solutioncontains 3-8 wt % concentrated NH₄OH in water and the sample is combinedwith the pretreatment solution in a ratio ranging from 2:3 to 2:1vol/vol.

Aspect D85. The method of any of aspects D82-D84, wherein the methodfurther comprises adding a washing solution to the sorbent after addingthe sample fluid and before adding the elution solution, the washingsolution comprising a basic compound and an organic solvent.

Aspect D86. The method of aspect D85, wherein the basic compound isselected from ammonium hydroxide (NH₄OH), NaOH, KOH, and combinationsthereof.

Aspect D87. The method of any of aspects D85-D86, wherein the organicsolvent is a protic organic solvent.

Aspect D88. The method of any of aspects D85-D86, wherein the organicsolvent is methanol.

Aspect D89. The method of aspect D85, wherein the washing solutioncomprises NH₄OH and methanol.

Aspect D90. The method of aspect D85, wherein the washing solutioncontains 3-8 wt % conc. NH₄OH (aq) in methanol.

Aspect D91. The method of any of aspects D82-D84, wherein the methodfurther comprises (a) adding a first washing solution to the sorbentafter adding the sample fluid and before adding the elution solution,the washing solution comprising a basic compound and water and (b)adding a second washing solution to the sorbent after adding the firstwashing fluid and before adding the elution solution, the second washingsolution comprising an organic solvent.

Aspect D92. The method of aspect D91, wherein the basic compound isselected from ammonium hydroxide (NH₄OH), NaOH, KOH, ammoniumbicarbonate, pyridine, piperazine, and combinations thereof.

Aspect D93. The method of aspect D91, wherein the basic compound isNH₄OH.

Aspect D94. The method of any of aspects D91-D93, wherein the organicsolvent is a protic organic solvent.

Aspect D95. The method of any of aspects D91-D93, wherein the organicsolvent is methanol.

Aspect D96. The method of aspect D91, wherein the first washing solutioncomprises NH₄OH in water and the second washing solution comprisesmethanol.

Aspect D97. The method of aspect D96, wherein the first washing solutioncontains 3-8 wt % conc. NH₄OH in water.

Aspect D98. The method of any of aspects D1-D97, wherein the sample isselected from a plasma sample and a serum sample.

Aspect E1. A kit comprising a sorbent that comprises a silica component,a C4-C60 alkyl component, and an organic copolymer component comprisinga hydrophobic organic monomer and a hydrophilic organic monomer and oneor more kit components selected from (a) an elution solution thatcomprises a protic solvent and a polar aprotic solvent (b) apretreatment solution, (c) one or more washing solutions, (d) acollection plate or barrel, (e) a cap mat, (f) calibration and referencestandards, and (g) Identification tagging for each component.

Aspect E2. The kit of aspect E1, wherein the sorbent is provided inmulti-well strip, a multi-well plate, a column, or a single-usecartridge.

Aspect E3. The kit of any of aspects E1-E2, wherein the elution solutioncomprises a polar aprotic solvent selected from acetonitrile, acetone,tetrahydrofuran, methylene chloride, ethyl acetate,N,N-dimethylformamide, dimethyl sulfoxide and dimethyl ether.

Aspect E4. The kit of any of aspects E1-E3, wherein the elution solutioncomprises a protic solvent selected from methanol, ethanol, 1-propanol,2-propanol, t-butanol, acetic acid, formic acid, aqueous ammonia,methanolic ammonia, water, and blends thereof.

Aspect E5. The kit of any of aspects E1-E2, wherein the elution solutioncomprises acetonitrile as a polar aprotic solvent and methanol as aprotic solvent.

Aspect E6. The kit of any of aspects E1-E5, wherein the elution solutioncomprises from 60 vol % to 100 vol % polar aprotic solvent and from 0vol % to 40 vol % protic solvent.

Aspect E7. The kit of any of aspects E1-E6, wherein the alkyl componentof the sorbent is bound to the silica component.

Aspect E8. The kit of any of aspects E1-E7, wherein the hydrophilicorganic monomer comprises a monomer having the following formula,

where n ranges from 1-3.

Aspect E9. The kit of any of aspects E1-E8, wherein the hydrophobicorganic monomer comprises divinylbenzene and, optionally, styrene.

Aspect E10. The kit of any of aspects E1-E9, wherein the sorbent is notan ion exchange sorbent.

Aspect E11. The kit of aspect E10, wherein the elution solutioncomprises acetonitrile and methanol in a ratio ranging from 70:30 to95:5 vol/vol.

Aspect E12. The kit of any of aspects E10-E11, wherein the pretreatmentsolution comprises a pretreatment compound, water, and an organicsolvent.

Aspect E13. The kit of aspect E12, wherein the organic solvent is aprotic organic solvent.

Aspect E14. The kit of any of aspects E12-E13, wherein the pretreatmentcompound is H₃PO₄, formic acid, acetic acid, sulfuric acid, or a blendthereof.

Aspect E15. The kit of aspect E12, wherein the pretreatment solutioncomprises H₃PO₄, water and methanol.

Aspect E16. The kit of aspect E12, wherein the pretreatment solutioncontains 3-5 wt % concentrated H₃PO₄ in water and methanol, which areprovided in a ratio ranging from 92:8 to 97:3 vol/vol.

Aspect E17. The kit of any of aspects E10-E11, wherein the pretreatmentsolution comprises a pretreatment compound and water.

Aspect E18. The kit of aspect E17, wherein the pretreatment solutioncomprises H₃PO₄ and water.

Aspect E19. The kit of aspect E17, wherein the pretreatment solutioncontains 3-5 wt % concentrated H₃PO₄ in water.

Aspect E20. The kit of any of aspects E17-E19, wherein the washingsolution comprises water and an organic solvent.

Aspect E21. The kit of aspect E20, wherein the washing solutioncomprises water and a protic organic solvent.

Aspect E22. The kit of aspect E20, wherein the washing solutioncomprises water and methanol.

Aspect E23. The kit of aspect E20, wherein the washing solutioncomprises water and methanol in a ratio ranging from 90:10 to 98:2.

Aspect E24. The kit of any of aspects E1-E9, wherein the sorbent is astrong cation exchange sorbent.

Aspect E25. The kit of aspect E24, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore sulfonate groups.

Aspect E26. The kit of aspect E24, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more sulfonate groups.

Aspect E27. The kit of any of aspects E24-E26, wherein the kit isconfigured to remove matrix molecules from a sample containing a basictarget analyte.

Aspect E28. The kit of any of aspects E1-E9, wherein the sorbent is aweak anion exchange sorbent.

Aspect E29. The kit of aspect E28, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore primary, secondary or tertiary amine groups.

Aspect E30. The kit of aspect E28, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more piperazine groups.

Aspect E31. The kit of any of aspects E28-E30, wherein the kit isconfigured to remove matrix molecules from a sample containing a strongacid target analyte.

Aspect E32. The kit of any of aspects E24-E31, wherein the elutionsolution comprises water and a basic compound.

Aspect E33. The kit of aspect E32, wherein the basic compound selectedfrom ammonium hydroxide (NH₄OH), NaOH, KOH, ammonium bicarbonate andcombinations thereof.

Aspect E34. The kit of any of aspects E24-E33, wherein the elutionsolution comprises acetonitrile and methanol in a ratio ranging from60:40 to 100:0 vol/vol.

Aspect E35. The kit of any of aspects E24-E34, wherein the elutionsolution comprises NH₄OH (aq), acetonitrile and methanol.

Aspect E36. The kit of any of aspects E24-E35, wherein elution solutioncomprises 3-8 wt % concentrated NH₄OH in water.

Aspect E37. The kit of any of aspects E24-E36, wherein the pretreatmentsolution comprising an organic solvent and one or more pretreatmentcompounds.

Aspect E38. The kit of aspect E37, wherein the organic solvent is aprotic organic solvent.

Aspect E39. The kit of aspect E37, wherein the protic organic solvent ismethanol.

Aspect E40. The kit of any of aspects E37-E39, wherein the one or morepretreatment compounds a selected from formic acid, carbonic acid,acetic acid.

Aspect E41. The kit of aspect E40, wherein the one or more pretreatmentcompounds further comprise H₃PO₄.

Aspect E42. The kit of aspect E37, wherein the pretreatment solutioncomprises methanol, formic acid and H₃PO₄.

Aspect E43. The kit of aspect E37, wherein the pretreatment solutioncontains 1-4 wt % concentrated H₃PO₄ in water and 1-4 wt % formic acidin methanol.

Aspect E44. The kit of any of aspects E24-E36, wherein the pretreatmentsolution comprises water and a pretreatment compound.

Aspect E45. The kit of aspect E44, wherein the pretreatment compound isselected from H₃PO₄, formic acid, acetic acid, sulfuric acid, or a blendthereof.

Aspect E46. The kit of aspect E44, wherein the pretreatment compound isH₃PO₄.

Aspect E47. The kit of aspect E44, wherein the pretreatment solutioncontains 2-8 wt % concentrated H₃PO₄ in water.

Aspect E48. The kit of any of aspects E44-E47, wherein the washingsolution comprises an acidic compound and an organic solvent.

Aspect E49. The kit of aspect E48, wherein the organic solvent is aprotic organic solvent.

Aspect E50. The kit of aspect E48, wherein the organic solvent ismethanol.

Aspect E51. The method any of aspects E48-E50, wherein the acidiccompound is selected from formic acid, carbonic acid, and acetic acid.

Aspect E52. The kit of aspect E48, wherein the washing solutioncomprises formic acid and methanol.

Aspect E53. The kit of aspect E48, wherein the washing solution contains1-4 wt % formic acid in methanol.

Aspect E54. The kit of any of aspects E44-E47, wherein the kit comprises(a) a first washing solution that comprises an acidic compound and waterand (b) a second washing solution that comprises an organic solvent.

Aspect E55. The kit of aspect E54, wherein the acidic compound isselected from formic acid, carbonic acid, and acetic acid.

Aspect E56. The kit of aspect E54, wherein the acidic compound is formicacid.

Aspect E57. The kit of any of aspects E54-E56, wherein the organicsolvent is a protic organic solvent.

Aspect E58. The kit of any of aspects E54-E56, wherein the organicsolvent is methanol.

Aspect E59. The kit of aspect E54, wherein the first washing solutioncomprises formic acid in water and the second washing solution comprisesmethanol.

Aspect E60. The kit of aspect E54, wherein the first washing solutioncontains 1-4 wt % formic acid in water and the second washing solutioncomprises methanol.

Aspect E61. The kit of any of aspects E1-E9, wherein the sorbent is astrong anion exchange sorbent.

Aspect E62. The kit of aspect E61, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore quaternary ammonium groups.

Aspect E63. The kit of aspect E61, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore —R₁N⁺R₂R₃R₄ groups, where R₁ is a C1-C8 alkylene group, and R₂, R₃and R₄ are C1-C8 alkyl groups and may be the same or different.

Aspect E64. The kit of aspect E61, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more —R₁N⁺R₂R₃R₄ groups, where R₁ is a C1-C8 alkylene group, and R₂,R₃ and R₄ are C1-C8 alkyl groups and may be the same or different.

Aspect E65. The kit of any of aspects E63-E64, wherein w=1, x=0, y=0 andz=3.

Aspect E66. The kit of any of aspects E61-E65, wherein the kit isconfigured to remove matrix molecules from a sample containing an acidictarget analyte.

Aspect E67. The kit of any of aspects E1-E9, wherein the sorbent is aweak cation exchange sorbent.

Aspect E68. The kit of aspect E67, wherein the organic copolymercomponent further comprises an organic monomer that comprises one ormore carboxyl groups.

Aspect E69. The kit of aspect E67, wherein the organic copolymercomponent further comprises a divinylbenzene monomer that comprises oneor more carboxyl groups.

Aspect E70. The kit of any of aspects E67-E69, wherein the kit isconfigured to remove matrix molecules from a sample containing a targetanalyte that is a strong base or a quaternary amine compound.

Aspect E71. The kit of any of aspects E61-E70, wherein the elutionsolution comprises water and an acidic compound.

Aspect E72. The kit of aspect E71, wherein the acidic compound isselected from formic acid, acetic acid, and carbonic acid.

Aspect E73. The kit of any of aspects E61-E72, wherein the elutionsolution comprises acetonitrile and methanol in a ratio ranging from65:35 to 85:15 vol/vol.

Aspect E74. The kit of any of aspects E61-E73, wherein the elutionsolution comprises formic acid, acetonitrile and methanol.

Aspect E75. The kit of any of aspects E61-E74, wherein elution solutioncomprises 1-4 wt % formic acid.

Aspect E76. The kit of any of aspects E61-E75, wherein the pretreatmentsolution comprises an organic solvent and a pretreatment compound.

Aspect E77. The kit of aspect E76, wherein the pretreatment compound isselected from NH₄OH, ammonium bicarbonate, pyridine, piperazine, andcombinations thereof.

Aspect E78. The kit of any of aspects E76-E77, wherein the organicsolvent is a protic organic solvent.

Aspect E79. The kit of any of aspects E76-E77, wherein the organicsolvent is methanol.

Aspect E80. The kit of aspect E76, wherein the pretreatment solutioncomprises NH₄OH in methanol.

Aspect E81. The kit of aspect E76, wherein the pretreatment solutioncontains 3-8 wt % concentrated NH₄OH (aq) in methanol

Aspect E82. The kit of any of aspects E61-E75, wherein the pretreatmentsolution comprises water and a pretreatment compound.

Aspect E83. The kit of aspect E82, wherein the pretreatment compound isselected from NH₄OH, ammonium bicarbonate, pyridine, piperazine, andcombinations thereof.

Aspect E84. The kit of aspect E82, wherein the pretreatment solutioncontains 3-8 wt % concentrated NH₄OH in water.

Aspect E85. The kit of any of aspects E82-E84, wherein the washingsolution comprising a basic compound and an organic solvent.

Aspect E86. The kit of aspect E85, wherein the basic compound isselected from ammonium hydroxide (NH₄OH), NaOH, KOH, and combinationsthereof.

Aspect E87. The kit of any of aspects E85-E86, wherein the organicsolvent is a protic organic solvent.

Aspect E88. The kit of any of aspects E85-E86, wherein the organicsolvent is methanol.

Aspect E89. The kit of aspect E85, wherein the washing solutioncomprises NH₄OH and methanol.

Aspect E90. The kit of aspect E85, wherein the washing solution contains3-8 wt % conc. NH₄OH (aq) in methanol.

Aspect E91. The kit of any of aspects E82-E84, wherein the kit comprises(a) a first washing solution comprising a basic compound and water and(b) a second washing solution comprising an organic solvent.

Aspect E92. The kit of aspect E91, wherein the basic compound isselected from ammonium hydroxide (NH₄OH), NaOH, KOH, ammoniumbicarbonate, pyridine, piperazine, and combinations thereof.

Aspect E93. The kit of aspect E91, wherein the basic compound is NH₄OH.

Aspect E94. The kit of any of aspects E91-E93, wherein the organicsolvent is a protic organic solvent.

Aspect E95. The kit of any of aspects E91-E93, wherein the organicsolvent is methanol.

Aspect E96. The kit of aspect E91, wherein the first washing solutioncomprises NH₄OH in water and the second washing solution comprisesmethanol.

Aspect E97. The kit of aspect E96, wherein the first washing solutioncontains 3-8 wt % conc. NH₄OH in water.

These and other aspects, as well as numerous embodiments and advantagesassociated with the methods, kits and sorbents, and devices described inthe present disclosure will become immediately apparent to those ofordinary skill in the art upon review of the detailed description andclaims to follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows total phospholipid content (TIC peak area) levels in elutefraction for Oasis MCX™ sorbent particles compared to particles inaccordance with the present disclosure, using a preexisting methodprotocol and a method protocol in accordance with the presentdisclosure.

FIG. 2 shows percent analyte recovery in elute fraction for Oasis MCX™sorbent particles compared to particles in accordance with the presentdisclosure, using a preexisting method protocol.

FIG. 3 shows total phospholipid content (TIC peak area) levels in elutefraction for Oasis MCX™ sorbent particles compared to particles inaccordance with the present disclosure, using a preexisting methodprotocol and a method protocol in accordance with the presentdisclosure.

FIG. 4 shows percent analyte recovery in elute fraction for Oasis MCX™sorbent particles compared to particles in accordance with the presentdisclosure, using a preexisting method protocol and a method protocol inaccordance with the present disclosure.

FIG. 5 shows specific reagent amounts and reaction conditions forExample 2.

FIG. 6 shows analytical data for the hybrid particles of Example 2a.

DETAILED DESCRIPTION

The present disclosure relates to sorbents, devices, kits and methodsthat may be used for treatment of biological samples, including removalof matrix molecules from a biological sample that contains (orpotentially contains) an analyte of interest, including removal of saltsand phospholipids in order to clean-up and/or concentrate analytes ofinterest by Solid Phase Extraction (SPE).

Sorbents

Sorbents in accordance with the present disclosure include sorbents thatcomprises (a) a silica component, (b) an organic copolymer componentcomprising at least one hydrophobic organic monomer and at least onehydrophilic organic monomer and, optionally, (c) a C4-C60 alkylcomponent, typically C4-C50 alkyl component, more typically a C8-C30alkyl component, and even more typically, a C12-C18 alkyl component. Inthis regard, the following description of various compounds havingC4-C60 alkyl components is to be understood as likewise a description ofthe same compounds having a C4-C50 alkyl components, a C8-C30 alkylcomponents, or a C12-C18 alkyl components. Moreover, the followingdescription of various compounds having a C4-C60 alkyl components is tobe understood to include a C4-C60 alkyl-silyl components.

Sorbents for use in the present disclosure may comprise sorbentparticles, which may vary widely in size and may range, for example,from about 5 to about 60 m in diameter, among other possible particlesizes. Sorbent particles for use in conjunction with the presentdisclosure may be porous or non-porous.

As used herein, “hydrophilic” means having an affinity for, attracting,adsorbing or absorbing water, and “hydrophobic” means lacking anaffinity for, repelling, or failing to adsorb or absorb water.

As used herein, “polymers” are molecules containing repeating subunits.

As used herein, “organic polymers” are organic molecules containingrepeating subunits.

The term “monomer”, as used herein, refers to both a molecule comprisingone or more polymerizable functional groups prior to polymerization, anda repeating subunit of a polymer. As a specific example, the followingpolymerizable monomer,

and the following incorporated monomer,

may be each be referred to herein as a “styrene sulfonate monomer,”regardless of when the sulfonate group is introduced (e.g., formed bypolymerization of

or formed by polymerization of styrene followed by sulfonation of theincorporated monomer) It is noted that incorporated monomers may benamed independently of the organic monomer that was polymerized to formthe polymer. For instance, in the preceding example, the incorporatedstyrene sulfonate monomer may, in theory, be formed from a free styrenesulfonate monomer or from a styrene monomer that is first polymerizedand later subjected to a sulfonation process. In either case, theincorporated monomer may be referred to herein as a “styrene sulfonatemonomer”.

Organic polymers may take on a number of configurations, which may beselected, for example, from linear, cyclic and branched configurations,among others. Branched configurations include star-shaped configurations(e.g., configurations in which three or more chains emanate from asingle branch region), comb configurations (e.g., configurations havinga polymer backbone and a plurality of side chains, also referred to as“graft” configurations), dendritic configurations (e.g., arborescent andhyperbranched polymers), networked (e.g., crosslinked) configurations,and so forth.

As used herein, organic polymers include “organic homopolymers,” whichare polymers that contain multiple copies of a single organic monomer,and “organic copolymers,” which are organic polymers that containmultiple copies of at least two dissimilar organic monomers, examples ofwhich include random, statistical, gradient, periodic (e.g.,alternating) and block copolymers, among others.

Sorbent Copolymer Component

With regard to the organic copolymer component of the sorbent thatcomprises at least one hydrophobic organic monomer and at least onehydrophilic organic monomer, in certain embodiments, the hydrophilicorganic monomer may be selected from organic monomers having an amidegroup, organic monomers having an ester group, organic monomers having acarbonate group, organic monomers having a carbamate group, organicmonomers having a urea group, organic monomers having a hydroxyl group,and organic monomers having nitrogen-containing heterocyclic group,among other possibilities. Specific examples of hydrophilic organicmonomers include, for example, 2-vinylpyridine, 3-vinylpyridine,4-vinylpyridine, N-vinylpyrrolidone, N-vinyl-piperidone, N-vinylcaprolactam, lower alkyl acrylates (e.g., methyl acrylate, ethylacrylate, etc.), lower alkyl methacrylates (e.g., methyl methacrylate,ethyl methacrylate, etc.), vinyl acetate, acrylamide or methacrylamide,hydroxypolyethoxy allyl ether, ethoxy ethyl methacrylate, ethyleneglycol dimethacrylate, or diallyl maleate. In certain beneficialembodiments, the hydrophilic organic monomer may be a monomer having thefollowing formula,

where n ranges from 1-3 (i.e., N-vinyl pyrrolidone,N-vinyl-2-piperidinone or N-vinyl caprolactam).

In certain embodiments, the hydrophobic monomer of the organic copolymercomponent may comprise a C6-C18 monocyclic or multicyclic carbocyclicgroup, e.g., a phenyl group or a phenylene group. Specific examples ofhydrophobic monomers include, for example, monofunctional andmultifunctional aromatic monomers such as styrene and divinylbenzene,monofunctional and multifunctional olefin monomers such as ethylene,propylene or butylene, polycarbonate monomers, ethylene terephthalate,monofunctional and multifunctional fluorinated monomers such asfluoroethylene, 1,1-difluoroethylene), tetrafluoroethylene,chlorotrifluoroethylene, hexafluoropropylene, perfluoropropylvinylether,or perfluoromethylvinylether, monofunctional or multifunctional acrylatemonomers having a higher alkyl group, monofunctional or multifunctionalacrylate monomers having a C6-C18 saturated, unsaturated or aromaticcarbocyclic group, monofunctional or multifunctional methacrylatemonomers having a higher alkyl group, monofunctional or multifunctionalmethacrylate monomers having a C6-C18 saturated, unsaturated or aromaticcarbocyclic group, among others. In certain embodiments, DVB 80 may beemployed, which is a monomer mixture that comprises divinylbenzene (80%)as well as a mixture of ethyl-styrene isomers, diethylbenzene, and caninclude other isomers as well.

In certain embodiments, the organic copolymer component will comprise amultifunctional hydrophobic organic monomer such as divinylbenzeneand/or a multifunctional hydrophilic organic monomer, such as ethyleneglycol dimethacrylate, methylene bisacrylamide or allyl methacrylate, inorder to provide crosslinks in the organic copolymer.

In certain embodiments, the organic copolymer component may comprisen-vinyl pyrrolidone or n-vinyl caprolactam as a hydrophilic organicmonomer and divinylbenzene as a hydrophobic organic monomer.

In certain embodiments, the organic copolymer may comprise an organicmonomer that comprises one or more anionic groups and/or an organicmonomer that comprises one or more cationic groups, for example, toprovide the sorbent with ion exchange characteristics.

The organic copolymer may comprise, for example, an organic monomer thatprovides strong cation exchange characteristics, in particular, anorganic monomer having one or more anionic groups that maintain anegative charge over a wide pH range such as, for instance, sulfonategroups. In particular embodiments, the organic monomer may be asulfonyl-substituted divinyl benzene monomer. Monomers having one ormore sulfonate groups may be incorporated in the copolymer, for example,by polymerizing the copolymer from a monomer mixture that includes asulfonated free monomer or by subjecting a previously formed copolymerto a sulfonation process, for instance, by exposing the previouslyformed copolymer to concentrated sulfuric acid. For example, incopolymers formed from divinyl benzene, previously polymerized divinylbenzene monomers may sulfonated in this fashion.

The organic copolymer may comprise, for example, an organic monomer thatprovides strong anion exchange characteristics, in particular, anorganic monomer having one or cationic groups that maintain a positivecharge over a wide pH range such as quaternary ammonium groups, forinstance, an organic monomer that comprises one or more —R₁—N⁺R₂R₃R₄groups, where R₁ is an alkylene group, typically, a C1-C8 alkylene group(i.e., selected from C1, C2, C3, C4, C5, C6, C7, and C8 alkylenegroups), and R₂, R₃ and R₄ may be the same or different and are alkylgroups, typically, C1-C8 alkyl groups (i.e., selected from C1, C2, C3,C4, C5, C6, C7, and C8 alkyl groups). As a specific example, afterformation of a copolymer that comprises divinylbenzene, thedivinylbenzene monomer within the copolymer can be chloromethylated,followed by amination as described, for example, in U.S. Pat. No.7,442,299, which is hereby incorporated by reference. In the presentcase, a tertiary amine may be used to form a quaternary amine uponreaction. In particular embodiments, the organic monomer may be aquaternary-ammonium-substituted divinyl benzene monomer.

The organic copolymer may comprise, for example, an organic monomer thatprovides weak cation ion exchange characteristics, in particular, anorganic monomer having one or more anionic groups that becomeneutralized at lower pH levels such as, for instance, carboxylategroups. As a specific example, after formation of a copolymer thatcomprises divinylbenzene, the divinylbenzene monomer within thecopolymer can be chloromethylated, followed by oxidation to formcarboxylic acid groups. In particular embodiments, the organic monomermay be a carboxyl-substituted divinyl benzene monomer.

The organic copolymer may comprise, for example, an organic monomer thatprovides weak anion exchange characteristics, in particular, an organicmonomer having one or cationic groups that become neutralized at higherpH levels such as, for instance, primary, secondary or tertiary aminegroups, for example, piperazinyl, N-methylpiperazinyl, pyrazinyl,piperidinyl, morpholino, pyrrolidinyl, quinolinyl, pyridyl, pyrimidyl,pyrrolyl, or indolyl groups or phosphate (3-) or carbonate (2-) groups.As a specific example, after formation of a copolymer that comprisesdivinylbenzene, the divinylbenzene monomer within the copolymer can bechloromethylated, followed by amination as described, for example, inU.S. Pat. No. 7,731,844, which is hereby incorporated by reference. Inparticular embodiments, the organic monomer may be apiperazinyl-substituted divinyl benzene monomer.

In certain embodiments, the organic copolymer may further comprise analkenyl-functionalized organosilane monomer, thereby providing theorganic copolymer with organosilane groups, as discussed in detailfurther below. Specific examples of alkenyl-functionalized organosilanemonomers include 3-(trimethoxysilyl)propyl methacrylate (also so knownas 3-methacryloxypropyltrimethoxysilane, or MAPTMOS,vinyltriethoxysilane (VTES), methacryloxypropyltriethoxysilane,vinyltrimethoxy silane,N-(3-acryloxy-2-hydroxypropyl)-3-aminopropyltriethoxysilane,(3-acryloxypropyl)trimethoxysilane,0-(methacryloxyethyl)-N-(triethoxysilylpropyl)urethane, N-(3-methacryloxy-2-hydroxypropyl)-3-aminopropyltricthoxysilane,methacryloxymethyltriethoxysilane, methacryloxymethyltrimethoxysilane,methacryloxypropylmethyldiethoxysilane,methacryloxypropylmethyldimethoxysilane,methacryloxypropyltris(methoxyethoxy)silane,3-(N-styrylmethyl-2-aminoethylamino)propyltrimethoxysilanehydrochloride, among others.

Sorbent Silica Component

With regard to the silica component of the sorbent, the silica mayeither be in the form of substantially pure silica (corresponding to thecase of n=0 in the formulas to follow) or may comprise material havingthe formula SiO₂/(R² _(p)R⁴ _(g)SiO_(t))_(n) or the formula SiO₂/[R⁶(R²_(r)SiO_(t))_(m)]_(n) wherein R² and R⁴ are independently C1-C18aliphatic or aromatic moieties, R⁶ is a substituted or unsubstitutedC1-C18 alkylene, alkenylene, alkynylene or arylene moiety bridging twoor more silicon atoms, p and q are 0, 1 or 2, provided that p+q=1 or 2,and that when p+q=1, t=1.5, and when p+q=2, t=1; r is 0 or 1, providedthat when r=0, t=1.5, and when r=1, t=1; m is an integer greater than orequal to 2, and n is a number from 0.03 or less to 1, for example,ranging from 0.03 to 0.1 to 0.2 to 0.3 to 0.4 to 0.5 to 0.6 to 0.7 to0.8 to 0.9 to 0.95 to 1) (i.e., ranging between any two of the precedingvalues).

In various embodiments, the silica component is formed by hydrolyticallycondensing one or more silane compounds, which typically include (a) oneor more silane compounds of the formula SiZ₁Z₂Z₃Z₄, where Z₁, Z₂, Z₃ andZ₄ are independently selected from Cl, Br, I, C1-C4 alkoxy, C1-C4alkylamino, and C1-C4 alkyl, although at most three of Z₁, Z₂, Z₃ and Z₄can be C1-C4 alkyl, for example, tetraalkoxysilanes, including,tetra-C1-C4-alkoxysilanes such as tetramethoxysilane, tetraethoxysilane,tetrachlorosilane, methyl-triethoxysilane, and methyl-trichlorosilane,among others, and alkyl-trialkoxysilanes, typicallyC1-C4-alkyl-tri-C1-C4-alkoxysilanes, such as methyl-triethoxysilane,among others. and/or (b) one or more compounds of the formula SiZ₁Z₂Z₃—R—SiZ₄Z₅Z₆, where Z₁, Z₂ and Z₃ are independently selected fromCl, Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C4 alkyl, although atmost two of Z₁, Z₂ and Z₃ can be C1-C4 alkyl, and where Z₄, Z₅ and Z₆are independently selected from Cl, Br, I, C1-C4 alkoxy, C1-C4alkylamino, and C1-C4 alkyl, although at most two of Z₄, Z₅ and Z₆ canbe C1-C4 alkyl, for example, bis(trialkoxysilyl)alkanes, typically,bis(tri-C1-C4-alkoxysilyl)C1-C4-alkanes such asbis(triethoxysilyl)ethane, among others. Numerous suitable organosilanecompounds are set forth in U.S. Pat. No. 6,686,035, the disclosure ofwhich is hereby incorporated by reference.

In certain embodiments, the silica component may include ahydrolytically condensed alkenyl-functionalized organosilane monomer,thereby providing the silica component with alkene groups from whichorganic polymerization can proceed. Specific examples ofalkenyl-functionalized organosilane monomers are listed above andinclude 3-(trimethoxysilyl)propyl methacrylate (MAPTMOS) andvinyltriethoxysilane (VTES), among others.

In embodiments where the sorbent is provided with a C4-C60 alkylcomponent, the silica component may include a hydrolytically condensedC4-C60-alkyl-functionalized organosilane compound, such as, for example,a compound of the formula RSiZ₁Z₂Z₃, where R is C4-C60 alkyl and whereZ₁, Z₂ and Z₃ are independently selected from Cl, Br, I, C1-C4 alkoxy,C1-C4 alkylamino, and C1-C4 alkyl, although at most two of Z₁, Z₂ and Z₃can be C1-C4 alkyl, specific examples of which include aC4-C60-alkyl-tri-C1-C3-alkoxysilane, a C4-C60-alkyl-trichlorosilane, aC4-C60-alkyl-tri-C1-C3-alkylaminosilane, aC4-C60-alkyl-C1-C3-alkoxydichlorosilane, aC4-C60-alkyl-C1-C3-alkyl-di-C1-C3-alkoxysilane, aC4-C60-alkyl-C1-C3-alkyl-dichlorosilane, aC4-C60-alkyl-C1-C3-dialkyl-C1-C3-alkoxysilane, and aC1-C3-dialkyl-C4-C60-alkylchlorosilane. Specific examples ofC4-C60-alkyl-functionalized organosilane compounds includeoctadecyltrimethoxysilane, octadecyltriethoxysilane,octadecyltrichlorosilane, octadecyltri(dimethylamino)silane,methyloctadecyldimethoxysilane, methyloctadecyldiethoxysilane,methyloctadecyldichlorosilane, methyloctadecyldi(dimethylamino)silane,dimethyloctadecylmethoxysilane, dimethyloctadecylethoxysilane,dimethyloctadecylchlorosilane, dimethyloctadecyldimethylaminosilane,diisopropyloctadecylmethoxysilane, diisopropyloctadecylethoxysilane,diisopropyloctadecylchlorosilane,diisopropyloctadecyldimethylaminosilane, octyltrimethoxysilane,octyltriethoxysilane, octyltrichlorosilane,octyltri(dimethylamino)silane, methyloctyldimethoxysilane,methyloctyldiethoxysilane, methyloctyldichlorosilane,methyloctyldi(dimethylamino)silane, dimethyloctylmethoxysilane,dimethyloctylethoxysilane, dimethyloctylchlorosilane,dimethyloctyldimethylaminosilane, diisopropyloctylmethoxysilane,diisopropyloctylethoxysilane, diisopropyloctylchlorosilane,diisopropyloctyldimethylaminosilane, butyltrimethoxysilane,butyltriethoxysilane, butyltrichlorosilane,butyltri(dimethylamino)silane, methylbutyldimethoxysilane,methylbutyldiethoxysilane, methylbutyldichlorosilane,methylbutyldi(dimethylamino)silane, dimethylbutylmethoxysilane,dimethylbutylethoxysilane, dimethylbutylchlorosilane,dimethylbutyldimethylaminosilane, diisopropylbutylmethoxysilane,diisopropylbutylethoxysilane, diisopropylbutylchlorosilane, anddiisopropylbutyldimethylaminosilane.

Sorbent Particles with Organic Copolymer Core Region and InorganicSurface Region

In certain embodiments, sorbent particles in accordance with the presentdisclosure include (a) a core region that comprises an organic copolymercomprising (i) at least one hydrophobic organic monomer, (ii) at leastone hydrophilic organic monomer, (iii) optionally, an organic monomerthat comprises one or more anionic groups and/or an organic monomer thatcomprises one or more cationic groups and/or an organic monomer thatcomprises one or more zwitterionic groups (where ion exchange capabilityis desired), and (iv) optionally, an alkenyl-functionalized organosilanemonomer, and (b) a surface region that comprises a silica component and,optionally, a C4-C60 alkyl component. The association of the one or morealkenyl-functionalized organosilane monomers with the organic copolymerof the core region provides organosilane groups which can furtherparticipate in hydrolytic condensation, which may in turn improvebonding between the core region and the surface region.

It is noted that, where the organic polymer comprises an organic monomerthat comprises one or more anionic groups, one or more cationic groupsand/or one or more zwitterionic groups, such charged groups may beintroduced during polymer formation by selecting a polymerizable monomerhaving such groups and/or such groups may be introduced after aprecursor organic polymer is formed via suitable chemical process. As aspecific example, monomers having one or more sulfonate groups may beformed in the copolymer, for example, by polymerizing the copolymer froma monomer mixture that includes a sulfonated free monomer (e.g., styrenesulfonate) or by subjecting a previously formed copolymer (e.g., anorganic copolymer comprising incorporated styrene monomers) to asulfonation process (e.g., sulfonating at least a portion of the styrenemonomers in the organic polymer to form incorporated styrene sulfonatemonomers).

Examples of hydrophobic organic monomers, hydrophilic organic monomers,organic monomers that comprise cationic groups, organic monomers thatcomprise anionic groups, and alkenyl-functionalized organosilanemonomers that may be used to form the core region are described above.Examples of organosilane compounds that may be hydrolytically condensedto form the surface region are also described above.

In various embodiments, the surface region may be formed byhydrolytically condensing one or more organosilane compounds at thesurface region of a previously formed particle core that comprises anorganic copolymer comprising (i) at least one hydrophobic organicmonomer, (ii) at least one hydrophilic organic monomer, (iii)optionally, an organic monomer that comprises one or more anionic groupsand/or an organic monomer that comprises one or more cationic groupsand/or an organic monomer that comprises one or more zwitterionicgroups, and (iv) optionally, an alkenyl-functionalized organosilanemonomer.

For instance, in some embodiments, the one or more organosilanecompounds that are hydrolytically condensed may comprise one or morealkoxysilane compounds, for example, one or more silane compounds of theformula SiZ₁Z₂Z₃Z₄, described above, among others, and optionally, aC4-C60-alkyl-functionalized organosilane compound, for example, of theformula RSiZ₁Z₂Z₃ described above, among others. Where aC4-C60-alkyl-functionalized organosilane compound is included, thiscompound may be hydrolytically condensed simultaneously with the one ormore alkoxysilane compounds, or the one or more alkoxysilane compoundsmay be hydrolytically condensed, followed by hydrolytic condensation ofthe C4-C60-alkyl-functionalized organosilane compound.

As indicated above, in some embodiments, one or morealkenyl-functionalized organosilane monomers, such as those discussedabove, among others, may be associated with the organic copolymer of thecore region. For example, the one or more alkenyl-functionalizedorganosilane monomers may be associated with the organic copolymer ofthe core region by copolymerization of the one or morealkenyl-functionalized organosilane monomers along with (i) the at leastone hydrophobic organic monomer, (ii) the at least one hydrophilicorganic monomer, and (iii) the optional organic monomer that comprisesone or more anionic groups and/or organic monomer that comprises one ormore cationic groups and/or an organic monomer that comprises one ormore zwitterionic groups.

As another example, the one or more alkenyl-functionalized organosilanemonomers may be associated with the organic copolymer of the core regionby first forming an organic copolymer that comprises the at least onehydrophobic organic monomer, the at least one hydrophilic organicmonomer, and the optional at least one organic monomer that comprisesone or more anionic groups and/or organic monomer that comprises one ormore cationic groups and/or an organic monomer that comprises one ormore zwitterionic groups, followed by reaction of the one or morealkenyl-functionalized organosilane monomers with the organic copolymer,for example, via residual unsaturation in the organic copolymer. Thisstep may be conducted after copolymerization and before core formationin some embodiments. This step may also be conducted aftercopolymerization and core formation in various embodiments.

Sorbent Particles with Inorganic Core Region and Organic CopolymerSurface Region

In certain embodiments, sorbent particles in accordance with the presentdisclosure may include (a) a core region that comprises a silicacomponent and (b) a surface region that comprises an organic copolymerhaving (i) at least one hydrophobic organic monomer (ii) at least onehydrophilic organic monomer and (iii) optionally, an organic monomerthat comprises one or more anionic groups and/or an organic monomer thatcomprises one or more cationic groups.

Particular examples of hydrophobic organic monomers, hydrophilic organicmonomers, organic monomers that comprise cationic groups, and organicmonomers that comprise anionic groups are provided above. Particularexamples of organic monomers that comprise zwitterionic groups can befound, for example, in André Laschewsky, “Structures and Synthesis ofZwitterionic Polymers,” Polymers 2014, 6(5), 1544-1601;doi:10.3390/polym6051544 and includeN-(2-methacryloyloxy)ethyl-N,N-dimethylammonio propanesulfonate (SPE),N-(3-methacryloylimino)propyl-N,N-dimethylammonio propanesulfonate(SPP), 2-(methacryloyloxy)ethylphosphatidylcholine (MPC), and3-(2′-vinyl-pyridinio)propanesulfonate (SPV), which are commerciallyavailable.

In certain embodiments, the core region is formed by hydrolyticallycondensing one or more silane compounds. Various silane compounds thatmay be hydrolytically condensed to form the core region, includingvarious alkoxysilane compounds, are described above.

In certain embodiments, the sorbent particles may further optionallyinclude a C4-C60 alkyl component. In those embodiments, the one or moresilane compounds that are hydrolytically condensed to form the coreregion may further comprise a C4-C60-alkyl-functionalized organosilanecompound, or one or more silane compounds that do not include aC4-C60-alkyl-functionalized organosilane compound may be hydrolyticallycondensed, followed by reaction with a C4-C60-alkyl-functionalizedorganosilane compound. Various C4-C60-alkyl-functionalized organosilanecompounds that may be hydrolytically condensed are described above.

In certain embodiments, the sorbent particles may be formed using analkenyl-functionalized organosilane species. For example, the one ormore silane compounds that are hydrolytically condensed to form the coreregion may further comprise an alkenyl-functionalized organosilanecompound. As another example, one or more silane compounds that do notinclude an alkenyl-functionalized organosilane may be hydrolyticallycondensed, followed by reaction with an alkenyl-functionalizedorganosilane compound. Various alkenyl-functionalized organosilanecompounds are described above. The resulting alkene groups at thesurface of the particle core may be used for forming a covalent bondbetween the particle core and the surface region containing the organicpolymer.

In some embodiments, the surface region that comprises the organiccopolymer is formed by a process that comprises an additionpolymerization of (i) at least one hydrophobic organic monomer (ii) atleast one hydrophilic organic monomer and (iii) optionally, an organicmonomer that comprises one or more anionic groups and/or an organicmonomer that comprises one or more cationic groups. Examples of additionpolymerization reactions include, for instance, anionic polymerizations,cationic polymerizations, or radical polymerization reactions, includingliving radical polymerization reactions such as ReversibleAddition-Fragmentation Chain Transfer Polymerization (RAFT), AtomTransfer Radical Polymerization (ATRP), and stable free radical (SFR)polymerizations (e.g., iniferter based polymerizations), among otherpossibilities.

In some embodiments, the surface region that comprises the organiccopolymer may be formed by a process that comprises forming the organiccopolymer and grafting the organic copolymer onto a surface of the coreregion (i.e., by a “grafting to” process). In some embodiments, thesurface region that comprises the organic copolymer may be formed by aprocess that comprises polymerizing the organic copolymer from sites onthe surface of the core region (by a “grafting from” process). Forinstance, where alkene groups are present at the surface of the coreregion, for example as a result of hydrolytic condensation of analkenyl-functionalized organosilane compound during the formation of thecore region, the alkene groups may be used as initiation sites for thepolymerization of the organic copolymer.

In embodiments where the sorbent particles further comprise aC4-C60-alkyl component, the organic copolymer may further comprise oneor more C4-C60 alkyl groups. For example, the organic monomerspolymerized to form the organic copolymer may further include aC4-C-60-alkyl functionalized unsaturated monomer, or aC4-C-60-alkyl-group may be attached to the organic copolymer.

Additional Sorbent Particles

In certain embodiments, sorbent particles in accordance with the presentdisclosure may comprise an organic copolymer that comprises (i) at leastone hydrophilic organic monomer, (ii) at least one hydrophobic organicmonomer, (iii) at least one alkenyl-functionalized organosilane monomer,and (iv) optionally, an organic monomer that comprises one or moreanionic groups and/or an organic monomer that comprises one or morecationic groups. Specific examples of hydrophobic organic monomers,hydrophilic organic monomers, organic monomers that comprise cationicgroups, organic monomers that comprise anionic groups, organic monomersthat comprise zwitterionic groups, and alkenyl-functionalizedorganosilane monomers that may be used to form the organic copolymer areprovided above.

The sorbent particles may be further modified by the addition of aC4-C60 alkyl component. For example, a C4-C60 alkyl component may beadded by hydrolytic condensation of a C4-C60-alkyl-functionalizedorganosilane compound. Various C4-C60-alkyl-functionalized organosilanecompounds that may be hydrolytically condensed are described above.

Devices

Devices in accordance with the present disclosure commonly includesorbents such as those described hereinabove in a packed sorbent bed.

Devices in accordance with the present disclosure commonly include ahousing having a chamber for accepting and holding sorbent. In variousembodiments, the housing maybe provided an inlet and an outlet. Theconstruction materials for the housing include inorganic materials, forinstance, metals such as stainless steel and ceramics such as glass, aswell as synthetic polymeric materials such as polyethylene,polypropylene, polyether ether ketone (PEEK), and polycarbonate, amongothers.

In certain embodiments, the device may include one or more filters whichact to hold the sorbent in a housing. Exemplary filters may be, forexample, in a form of membrane, screen, frit or spherical porous filter.

In certain embodiments, a solution received in the housing may flow intothe sorbent spontaneously, for example, capillary action. Alternatively,the flow may be generated through the sorbent by external forces, suchas gravity or centrifugation, or by applying a vacuum to an outlet ofthe housing or positive pressure to an inlet of the housing.

Specific examples of housings for use in the present disclosure include,for example, a syringe, an injection cartridge, a column (e.g., amicrobore column, capillary column or nanocolumn), a multi-well devicesuch as a 4 to 8-well rack, a 4 to 8-well strip, a 48 to 96-well plate,a 96 to 384-well micro-elution plate, micro-elution tip devices,including a 4 to 8-tip micro-elution strip, a 96 to 384-micro-elutiontip array, a single micro-elution pipet tip, a thin layer plate, amicrotiter plate, a spin tube, or a spin container, among others.

Multi-well formats are commonly used with robotic fluid dispensingsystems. Typical multi-well formats include 48-, 96-, and 384-wellstandard plate formats, although other formats are clearly possible.

Methods

As previously noted, the present disclosure also pertains to methodsthat may be used for removal of matrix molecules from a sample thatcontains an analyte of interest, including the removal phospholipids toclean-up and/or concentrate analytes of interest by Solid PhaseExtraction (SPE). Such methods can be used in conjunction with variouscommercially available sorbents, including Oasis HLB™ sorbent particles,Oasis MCX™ sorbent particles, Oasis WCX™ sorbent particles, Oasis MAX™sorbent particles and Oasis WAX™ sorbent particles, as well as thevarious sorbents described elsewhere herein, among others.

In various aspects, methods of performing a sample treatment procedureare provided which comprise: (a) adding a sample fluid comprising asample that comprises at least one target analyte to a sorbent thatcomprises an organic copolymer component comprising at least onehydrophobic organic monomer and at least one hydrophilic organicmonomer, a silica component, and a C4-C60 alkyl component, therebyyielding a sorbent with bound target analyte and (b) adding an elutionsolution that comprises a protic solvent and a polar aprotic solvent tothe sorbent, thereby desorbing the target analyte from the sorbent andforming a solution of the target analyte in the elution fluid.

In various embodiments, a sample fluid that comprises at least onetarget analyte and one or more phospholipids is added to the sorbent,thereby yielding a sorbent with bound target analyte and boundphospholipids, with subsequent addition of the elution solutionresulting in desorption of the target analyte from the sorbent, therebyforming a solution of the target analyte in the elution fluid, while atthe same time retaining at least 50% of the phospholipids on thesorbent, preferably, 70-100% of the phospholipids on the sorbent, morepreferably 85-100% of the phospholipids on the sorbent, and even morepreferably 90-100% of the phospholipids on the sorbent,

Particular examples of sorbents are described above, although anysuitable sorbent that comprises (a) an organic copolymer componentcomprising at least one hydrophobic organic monomer and at least onehydrophilic organic monomer, (b) a silica component, and (c) a C4-C60alkyl component may be employed.

Particular examples of organic copolymer components, includingparticular examples of hydrophobic organic monomers and hydrophilicorganic monomers, particular examples of silica components, andparticular examples of C4-C60 alkyl components are described above.

In various embodiments, the sample fluid is or is derived from abiological sample. Exemplary biological samples include any biologicalsample that contains or potentially contains phospholipids, such asbiological fluids (e.g., whole blood samples, blood plasma samples,serum samples, oral fluids, urine, etc.), biological tissues, biologicalmatrices, cells (e.g., one or more types of cells), cell culturesupernatants, foods that contain phospholipids (e.g., meats, wholegrains, legumes, eggs, etc.), and food extracts.

As indicated above, the methods of performing sample enrichmentdescribed herein include a step where a sample fluid that comprises orpotentially comprises at least one target analyte is added to a sorbent.Such a process frequently referred to as a “loading” process, andcommonly involves passing the sample fluid through a device thatcontains the sorbent (such devices are described in more detail below).During loading, the one or more target analytes, if present, along withvarious non-target substances, including one or more phospholipids, areadsorbed onto the sorbent. Contact time and/or flow rate may beoptimized for proper diffusion kinetics and binding of the one or moretarget analytes and the one or more phospholipids to the sorbent. Ifdesired the sample fluid may be passed multiple times through thedevice.

In certain embodiments, for example, where the sorbent is not readilywater-wettable, conditioning and equilibrating steps may be performedprior to sample loading. For example, in an exemplary embodiment, theconditioning may be performed with methanol, ethanol, propanol,isopropyl alcohol, water and a mixture thereof, among otherpossibilities. For example, in an exemplary embodiment, theequilibrating solution may have similar or same ionic strength as thesample fluid to provide equilibrated partitioning environments in thesorbents when the sample fluid is loaded. Exemplary equilibratingsolution/solvent include, for example, water, an aqueous solution suchas a buffer solution (e.g., a phosphate buffer solution), awater-miscible organic solvent solution and the like. In certainembodiments, for example, where the sorbent is sufficientlywater-wettable, conditioning and equilibrating may be omitted beforesample loading.

In various embodiments, the elution solutions used in accordance withthe present disclosure may comprise (a) from 60 vol % to 100 vol % polaraprotic solvent, for example, ranging from 60 vol % to 65 vol % to 70vol % to 75 vol % to 80 vol % to 85 vol % to 90 vol % to 95 vol % to 98vol % to 100 vol % polar aprotic solvent and (b) from 0 vol % to 40 vol% protic solvent, for example ranging from 0 vol % to 2 vol % to 5 vol %to 10 vol % to 15 vol % to 20 vol % to 25 vol % to 30 vol % to 35 vol %to 40 vol % protic solvent. In certain embodiments, the elution solutionmay comprise 65-95 vol % of the polar aprotic solvent and 5-35 vol % ofthe protic solvent, more typically, 70-90 vol % of the polar aproticsolvent and 10-30 vol % of the protic solvent

Particular examples of protic solvents for use in conjunction with thepresent disclosure include, for example, methanol, ethanol, 1-propanol,2-propanol, t-butanol, acetic acid, formic acid, aqueous ammonia,methanolic ammonia, water, and blends thereof.

Particular examples of polar aprotic solvents for use in conjunctionwith the present disclosure include, for example, acetonitrile, acetone,tetrahydrofuran, methylene chloride, ethyl acetate,N,N-dimethylformamide, dimethyl sulfoxide, dimethyl ether, and blendsthereof.

The particular methods employed herein may vary based on the nature ofthe sorbent, specifically, whether the sorbent is an ion exchangesorbent (e.g., a strong cation exchange sorbent, a strong anion exchangesorbent, a weak cation exchange sorbent, or a weak anion exchangesorbent), or whether the sorbent is not an ion exchange sorbent.

Methods Based on Sorbents without Ion Exchange Functionality

In certain embodiments, sorbents may be employed which do not have ionexchange functionality.

In these embodiments, an elution solution like that described above maybe employed. For example, the elution solution may comprise 65-95 vol %of the polar aprotic solvent and 5-35 vol % of the protic solvent, moretypically 70-90 vol % of the polar aprotic solvent and 10-30 vol % ofthe protic solvent.

In these embodiments, the sample fluid may be prepared by combining asample with a pretreatment solution that comprises a pretreatmentcompound and water. In these embodiments, the sample may be combinedwith the pretreatment solution in a ratio ranging from 2:3 to 2:1vol/vol, among other possibilities.

In some embodiments, the pretreatment compound may be an acidiccompound, which may be selected from a wide variety of acidic compounds,including one or more of the following: organic acids such as formicacid, acetic acid, propionic acid, butyric acid, oxalic acid, malonicacid, succinic acid and maleic acid, among others and including organichydroxyacids such as glycolic acid, lactic acid, tartaric acid, malicacid, citric acid and gluconic acid, among others; and inorganic acidssuch as phosphoric acid, hydrochloric acid, nitric acid, sulfuric acid,or carbonic acid, among others. In certain beneficial embodiments, theacidic compound is selected from phosphoric acid, formic acid, aceticacid, sulfuric acid and blends thereof.

In particular embodiments, the pretreatment solution may comprise H₃PO₄and water. For example, the pretreatment solution may contain 3-5 wt %concentrated H₃PO₄ in water.

In some embodiments, the pretreatment solution may further comprise anorganic solvent in addition to the pretreatment compound and water.Where included, the organic solvent is typically a protic organicsolvent, which may be selected from those listed above, among others. Inthese embodiments, the sample may be combined with the pretreatmentsolution in a ratio ranging from 1:3 to 1:5 vol/vol, among otherpossibilities.

In particular embodiments, the pretreatment solution may comprise H₃PO₄,water and methanol. For example, the pretreatment solution may contain3-5 wt % concentrated H₃PO₄ in water combined with methanol, which areprovided in a ratio ranging from 92:8 to 97:3 vol/vol.

In various embodiments, the method may further comprise adding a washingsolution to the sorbent after adding the sample fluid and before addingthe elution solution. The washing solution may comprise, for example,water and an organic solvent, typically a protic organic solvent such asmethanol, among others. The water and protic organic solvent may becombined in a ratio ranging from 90:10 to 98:2 vol/vol, among otherpossibilities.

Specific embodiments of the present disclosure where sorbents are usedthat do not have ion exchange functionality are provided below in Table.1.

TABLE 1 2-Step Protocol 3-Step Protocol Step 1-Load Step 1-LoadPre-treated sample (e.g. Pre-treated sample dilute plasma 1:4 with (e.g.dilute plasma 1:1 4% H₃P0₄ in 94/6 with 4% H₃P0₄(aq)) water/methanol)Step 2-Wash 95/5 Water/Methanol Step 2-Elute Step 3-Elute 90/10 90/10Acetonitrile/Methanol Acetonitrile/Methanol

Methods Based on Strong Cation Exchange and Weak Anion Exchange Sorbents

In certain embodiments, a sorbent may be employed which is a strongcation exchange sorbent. Such sorbents may be employed, for example,where the target analyte is a basic target analyte (e.g., having a pKaof 2-10). In such embodiments, the target analyte is generally ionizedby the pre-treatment solution.

Examples of strong cation exchange sorbent include those wherein theorganic copolymer component comprises an organic monomer that comprisesone or more sulfonate groups. For example, the organic copolymercomponent may comprise a divinylbenzene monomer that comprises one ormore sulfonate groups, among other possibilities.

In certain embodiments, a sorbent may be employed which is a weak anionexchange sorbent which can be protonated in the pH range of 4 to 10.

Examples of weak anion exchange sorbents include those wherein theorganic copolymer component comprises an organic monomer that comprisesone or more primary, secondary or tertiary amine groups. For example,the organic copolymer component may comprise a divinylbenzene monomerthat comprises one or more piperazine groups, among other possibilities.

Where the sorbent is a strong cation exchange sorbent or a weak anionexchange sorbent, an elution solution like that described above, alongwith a basic compound may be employed. In certain embodiments, theelution solution may comprise 65-95 vol % of the polar aprotic solvent,5-35 vol % of the protic solvent and the basic compound (which istypically provided an amount that is less than or equal to thesaturation capacity of the sorbent), more specifically, 70-90 vol % ofthe polar aprotic solvent, 10-30 vol % of the protic solvent, and thebasic compound.

Examples of basic compounds may be selected from one or more of thefollowing, among others: ammonium hydroxide (NH₄OH), alkali metalhydroxides (e.g., NaOH, KOH, LiOH, RbOH, etc.), alkaline earth metalhydroxides (e.g., Mg(OH)₂, Ca(OH)₂, Sr(OH)₂, etc.), ammoniumbicarbonate, organic bases, and combinations thereof. In some beneficialembodiments, the basic compound may be selected from ammonium hydroxide(NH₄OH), NaOH, KOH, ammonium bicarbonate and combinations thereof.

In certain embodiments, the elution solution may comprise acetonitrileas a polar aprotic solvent, methanol as a protic solvent and NH₄OH as abase, for example, containing 3-8 wt % concentrated NH₄OH.

Where the sorbent is a strong cation exchange sorbent or a weak anionexchange sorbent, the sample fluid may comprise a sample of interestcombined with a pretreatment solution.

In some embodiments, the sample of interest is combined with apretreatment solution that contains an organic solvent and one or morepretreatment compounds. In certain beneficial embodiments, the organicsolvent is methanol.

Typically, the one or more pretreatment compounds include one or moreacidic pretreatment compounds. Suitable acidic compounds may be selectedfrom those listed above. In certain beneficial embodiments, the acidicpretreatment component may be selected from formic acid, carbonic acid,acetic acid, H₃PO₄, sulfuric acid, or a blend thereof.

In certain of these embodiments, the sample may be combined with thepretreatment solution in a ratio ranging from 2:3 to 2:1 vol/vol, amongother possibilities.

In certain embodiments, the pretreatment solution may comprise methanol,formic acid and H₃PO₄, for example, 1-4 wt % concentrated H₃PO₄ and 1-4wt % formic acid, in methanol.

In some embodiments, the sample of interest is combined with apretreatment solution that contains water and one or more pretreatmentcompounds.

The one or more pretreatment compounds may be acidic compounds which maybe selected from one or more acidic compounds such as those set forthabove, among others. As a specific example, the pretreatment solutionmay contain 2-8 wt % concentrated H₃PO₄ in water.

In some embodiments where the sample fluid contains a sample of interestcombined with a pretreatment solution that contains water and one ormore pretreatment compounds, the method may further comprise adding awashing solution to the sorbent after adding the sample fluid and beforeadding the elution solution. In these embodiments, the washing solutionmay comprise an acidic compound and an organic solvent, typically aprotic organic solvent. The acidic compound and the protic organicsolvent may be selected from those listed above. In certain embodiments,the acidic compound is selected from formic acid, carbonic acid, andacetic acid and the protic solvent may be methanol. In certainembodiments, the washing solution may comprise formic acid and methanol,for example, 1-4 wt % formic acid in methanol.

In other embodiments where the sample fluid contains a sample ofinterest combined with a pretreatment solution that contains water andone or more pretreatment compounds, the method may further comprise (a)adding a first washing solution to the sorbent after adding the samplefluid and before adding the elution solution, the first washing solutioncomprising an acidic compound and water and (b) adding a second washingsolution to the sorbent after adding the first washing fluid and beforeadding the elution solution, the second washing solution comprising anorganic solvent, typically a protic organic solvent. The acidic compoundfor the first washing solution may be selected from those listed above.In certain embodiment, the acidic compound is formic acid and theorganic solvent is methanol. For example, the first washing solution maycontain 1-4 wt % formic acid in water and the second washing solution bemethanol.

Specific embodiments of the present disclosure where a strong cationexchange sorbent or a weak anion exchange sorbent is employed as asorbent are provided below in Table 2.

2-Step Protocol 3-Step Protocol 4-Step Protocol Step 1-Load Step 1-LoadStep 1-Load Pre-treated Pre-treated Pre-treated sample (e.g. sample(e.g. sample (e.g. dilute plasma dilute plasma dilute plasma 1:1 1:4with 2% 1:1 with 4% with 4% H₃P0₄ and H₃P0₄(aq)) H₃P0₄(aq)) 2% FormicAcid in Methanol) Step 2-Wash Step 2-Wash 1 2% Formic Acid in 2% FormicAcid Methanol in water Step 3-Wash 2 100% Methanol Step 2-Elute Step3-Elute Step 4-Elute 5% NH₄OH in 5% NH₄OH in 5% NH₄OH in 75/25 (v/v)75/25 (v/v) 75/25 (v/v) Acetonitrile/ Acetonitrile/ Acetonitrile/Methanol Methanol Methanol

Methods Based on Strong Anion Exchange and Weak Cation Exchange Sorbents

In certain embodiments, a sorbent may be employed which is a stronganion exchange sorbent. Such sorbents may be employed, for example,where the target analyte is an acidic target analyte (e.g., having a pKaof 2-8)

Examples of strong anion exchange sorbent include those wherein theorganic copolymer component comprises an organic monomer that comprisesone or more quaternary ammonium groups. For example, the organiccopolymer component may comprise an organic monomer that comprises oneor more —R₁—N⁺R₂R₃R₄ groups, where R₁ is an alkylene group, typically, aC1-C8 alkylene group, and R₂, R₃ and R₄ may be the same or different andare alkyl groups, typically, C1-C8 alkyl groups. More particularly, theorganic copolymer component may further comprise a divinylbenzenemonomer that is substituted with one or more —R₁—N⁺R₂R₃R₄ groups.

In certain embodiments, a sorbent may be employed which is a weak cationexchange sorbent, which can be deprotonated in the pH range of 1-7.

Examples of weak cation exchange sorbents include those wherein theorganic copolymer component further comprises an organic monomer thatcomprises one or more carboxyl groups, for example, a divinylbenzenemonomer that comprises one or more carboxyl groups.

Where the sorbent is a strong anion exchange sorbent or a weak cationexchange sorbent, an elution solution like that described above, alongwith an acidic compound may be employed. In certain embodiments, theelution solution may comprise 65-95 vol % of the polar aprotic solvent,5-35 vol % of the protic solvent and the acidic compound (e.g., in anamount up to the sorbent capacity), typically 65-85 vol % of the polaraprotic solvent, and 15-35 vol % of the protic solvent, and the acidiccompound.

Examples of acidic compounds may be selected from one or more of thoselisted above, among others. In certain embodiments, the acidic compoundmay be an organic acid, for example, selected from formic acid, aceticacid, carbonic acid, and blends thereof.

In particular embodiments, the elution solution may compriseacetonitrile as a polar aprotic solvent, methanol as a protic solvent,and formic acid as an acidic compound, for example, containing 1-4 wt %formic acid.

Where the sorbent is a strong anion exchange sorbent or a weak cationexchange sorbent, the sample fluid may comprise a sample of interestcombined with a pretreatment solution.

In some embodiments, the sample of interest is combined with apretreatment solution that contains an organic solvent at highconcentration and one or more pretreatment compounds.

Typically, the organic solvent is a protic organic solvent, which may beselected from those set forth above, among others. In certain beneficialembodiments, the organic solvent is methanol.

Typically, the one or more pretreatment compounds include one or morebasic pretreatment compounds. Suitable basic pretreatment compounds maybe selected from a variety of basic compounds, including one or more ofthose basic compounds listed above. In certain beneficial embodiments,the pretreatment compound is selected from NH₄OH, ammonium bicarbonate,pyridine, piperazine, and combinations thereof.

In certain of these embodiments, the sample may be combined with thepretreatment solution in a ratio ranging from 1:2 to 1:8 vol/vol, amongother possibilities

In certain embodiments, the pretreatment solution may comprise methanoland NH₄OH, for example, 3-8 wt % concentrated NH₄OH in methanol.

In some embodiments, the sample of interest is combined with apretreatment solution that contains water and one or more pretreatmentcompounds.

The one or more pretreatment compounds may be basic compounds which maybe selected from one or more basic compounds such as those set forthabove, among others, including NH₄OH, ammonium bicarbonate, pyridine,piperazine, and combinations thereof.

In certain of these embodiments, the sample may be combined with thepretreatment solution in a ratio ranging from 2:3 to 2:1 vol/vol, amongother possibilities.

As a specific example, the pretreatment solution may contain 3-8 wt %concentrated NH₄OH in water.

In some embodiments where the sample fluid contains a sample of interestcombined with a pretreatment solution that contains water and one ormore pretreatment compounds, the method may further comprise adding awashing solution to the sorbent after adding the sample fluid and beforeadding the elution solution. In these embodiments, the washing solutionmay comprise a basic compound and an organic solvent, typically a proticorganic solvent, at high concentration. The basic compound and theprotic organic solvent may be selected from those listed above. Incertain embodiments, the basic compound may be selected from ammoniumhydroxide (NH₄OH), NaOH, KOH, and combinations thereof, and the proticsolvent may be methanol. In certain embodiments, the washing solutionmay comprise NH₄OH and methanol, for example, 3-8 wt % conc. NH₄OH inmethanol.

In other embodiments where the sample fluid contains a sample ofinterest combined with a pretreatment solution that contains water andone or more pretreatment compounds, the method may further comprise (a)adding a first washing solution to the sorbent after adding the samplefluid and before adding the elution solution, the first washing solutioncomprising a basic compound and water and (b) adding a second washingsolution to the sorbent after adding the first washing fluid and beforeadding the elution solution, the second washing solution comprising anorganic solvent, typically a protic organic solvent at highconcentration. The basic compound for the first washing solution may beselected from those listed above. In certain embodiment, the basiccompound is selected from ammonium hydroxide (NH₄OH), NaOH, KOH,ammonium bicarbonate, pyridine, piperazine, and combinations thereof. Incertain embodiments, the basic compound is NH₄OH and the organic solventis methanol. For example, the first washing solution may contain 3-8 wt% conc. NH₄OH in water and the second washing solution may be methanol.

Specific embodiments of the present disclosure where a strong anionexchange sorbent or a weak cation exchange sorbent is employed as asorbent are provided below in Table 3.

TABLE 3 2-Step Protocol 3-Step Protocol 4-Step Protocol Step 1-Load Step1-Load Step 1-Load Pre-treated sample Pre-treated sample Pre-treated(e.g. dilute (e.g. dilute sample (e.g. plasma 1:4 with plasma 1:1 with5% dilute plasma 5% NH₄OH in NH₄OH(aq)) 1:1 with methanol) 5% NH₄OH(aq))Step 2-Wash Step 2-Wash 1 5% NH₄OH in Methanol 5% NH₄OH (aq) Step 3-Wash2 100% Methanol Step 2-Elute Step 3-Elute Step 4-Elute 2% Formic Acid 2%Formic Acid 2% Formic Acid in in 75/25 (v/v) in 75/25 (v/v) 75/25 (v/v)Acetonitrile/Methanol Acetonitrile/Methanol Acetonitrile/Methanol

Additional Processing

After being eluted from the sorbent, further processing of the one ormore target analytes may include, for example, identifying, quantifying,or otherwise processing the one or more target analytes.

In various embodiments, methods described herein may further compriseanalyzing samples using analytical instruments and/or techniques, forexample, liquid chromatography (LC), including high performance liquidchromatography (HPLC) and ultra-high performance liquid chromatography(UHPLC), mass spectrometry (MS), including electrospray ionization massspectrometry (ESI-MS), matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS), time-of-flight mass spectrometry (TOFMS),nuclear magnetic resonance, infrared analysis, ultraviolet analysis, ora combination thereof. For instance, in some embodiments, the processedsamples may be analyzed using liquid chromatography, such as HPLC orUHPLC, in combination with mass spectrometry, such as MALDI-MS orESI-MS, examples of which include liquid chromatography-massspectrometry (LC-MS) techniques and liquid chromatography-massspectrometry/mass spectrometry (LC-MS/MS) techniques. In certainembodiments, the methods described herein are useful for direct to MSdetection, including direct analysis in real time (DART) massspectrometry and atmospheric solids analysis probe (ASAP) massspectrometry. In certain cases, analytical samples may be evaporated todryness, and then reconstituted in another solution before beinginjected into a liquid chromatography system.

Kits

Kits may also be provided which comprises a sorbent as describedelsewhere herein and one or more kit components selected from thefollowing: (a) an elution solution that comprises a protic solvent and apolar aprotic solvent, for example, selected from those elutionsolutions described hereinabove, (b) a pretreatment solution, forexample, selected from those pretreatment solutions describedhereinabove, (c) one or more washing solutions, for example, selectedfrom those washing solutions described hereinabove, (d) a collectionplate or barrel, (e) a cap mat, (f) calibration and reference standards,and (g) Identification tagging for each component, which may includepassive tags, such as RFID tags, for tracking the components. In certainembodiments, the sorbent may be provided in multi-well strip, amulti-well plate, a column, or a single-use cartridge as describedabove.

Example 1

Oasis MCX™ particles (Commercially available from Waters Corporation,Milford, Mass., USA) were fully dispersed in toluene (18 mL/g) thenazeotropically stripped (111° C., 1 h) to remove residual water from thematerial. These initial anhydrous conditions were important as theyensured water content accuracy when water was added in the subsequentstep. The reaction was cooled to 40° C. and held at that temperaturewhile a partially condensed polyethoxyoligosiloxane polymer (PEOS)consisting of 1:2:8n-octadecyltrimethoxysilane/1,2-bis(triethoxylsilyl)ethane(BTEE)/Tetraethyl orthosilicate (TEOS) was added (4.11 g PEOS/gparticles) was added and allowed to stir for 10 minutes followed bywater in the form of an acid or base catalyst, in this case NH₄OH(a_(q)) (0.125 g base/g particles). The reaction was stirred for anadditional 10 minutes at 40° C. before the temperature was increased to60° C. for 2 h. The reaction was then cooled to RT and the particleswere isolated via filtration. The particles were subsequently washedtwice with ethanol (10 ml/g) then redispersed in 70/30 (v/v)water/ethanol (10 mL/g). Ammonium Hydroxide (1 g NH₄OH/g particles) wasthen added and the mixture was stirred at 50° C. for 2 h. The reactionwas then cooled <40° C. and the particles were isolated via filtration.The isolated particles were washed (10 ml/g) using the followingsequence: 2× methanol/water (1:1 v/v), 2× methanol. Finally, theisolated, surface modified particles were dried for 16 h at 80° C. under25 mm vacuum.

Example 1A

To ensure uniformity of the hybrid layer, the modified particles fromExample 1 were exposed to elevated temperatures (100-140° C.) and pH(8-9.8) following the hydrothermal treatment process described in U.S.Pat. Nos. 6,686,035, 7,223,473 and 7,919,177, and in PCT applicationnumber WO 2008/103423.

Example 1B

Oasis MCX™ particles (Commercially available—Waters Corporation,Milford, Mass., USA) are surface modified using the process described inbelow.

Oasis MCX™ particles are fully dispersed in toluene (18 mL/g). Anazeotropic strip (111° C., 1 h) is then performed to remove residualwater from the material. These initial anhydrous conditions areimportant as they ensure water content accuracy when water is added inthe subsequent step. The reaction temperature is held at 40° C. while1:2:8 n-octadecyltrimethoxysilane/1,2-bis(triethoxylsilyl)ethane(BTEE)/Tetraethyl orthosilicate (TEOS) partially condensedpolyethoxyoligosiloxane polymer (PEOS) (4.11 g PEOS/g particles)(0.80-5.0 g PEOS/g cores) is added and allowed to stir for 10 minutesfollowed by water in the form of a catalyst (0.03-0.3 g acid or base/gcores). The reaction is stirred for an additional 10 minutes at 40° C.before the temperature is increased to 60° C. for 2 h. The reaction isthen cooled to RT and the particles are isolated via filtration. Theparticles are subsequently washed twice with ethanol (10 ml/g) thenredispersed in 70/30 (v/v) water/ethanol (10 mL/g). Ammonium Hydroxide(1 g NH₄OH/g cores) is added and the mixture is stirred at 50° C. for 2h. The reaction is then cooled <40° C. and the particles are isolatedvia filtration. The isolated particles are washed (10 ml/g) using thefollowing sequence: 2× methanol/water (1:1 v/v), 2× methanol. Finally,the isolated, surface modified particles are dried for 16 h at 80° C.under 25 mm vacuum. The above process is repeated 1-60 times.

Example 1C

To ensure uniformity of the hybrid layer, the modified particles fromExample 1B are exposed to elevated temperatures (100-140° C.) and pH(8-9.8) following the hydrothermal treatment process as noted in U.S.Pat. Nos. 6,686,035, 7,223,473 and 7,919,177, and in PCT applicationnumber WO 2008/103423. The process of attaching 100% BTEE PEOScore-coating material, as described above, is repeated 0-60 additionaltimes with or without subsequent exposure to elevated temperatures(100-140° C.) and pH (8-9.8) following the hydrothermal treatmentprocess as noted by Jiang {U.S. Pat. Nos. 6,686,035; 7,223,473;7,919,177} and Wyndham {WO 2008/103423}.

Example 1D

The process as described in Example 1B is further expanded to includemonomer and polymer inorganic and/or organic materials in place of 1:2:8n-octadecyltrimethoxysilane/1,2-bis(triethoxylsilyl)ethane(BTEE)/Tetraethyl orthosilicate (TEOS) PEOS.

Example 1E

As an alternative approach to producing the described product,cross-linking hybrid monomer such as but not limited to,methacryloxypropyltrimethoxysilane (MAPTMOS), Vinyltriethoxysilane(VTES), or others as listed in U.S. Pat. No. 8,791,220 may beincorporated onto the Oasis MCX™ particle surface, followed by theaddition of a hydrophobic alkyl phase via toluene reflux using a basecatalyst.

Example 2

Inorganic/organic hybrid particles used are produced as described inU.S. Pat. Nos. 8,791,220 and 9,211,524.

A solution of poly(vinyl alcohol) (PVA; 87%-89% hydrolyzed; Ave MW13,000-23,000) in water was prepared by mixing and heating to 80° C. for0.5 hours. Upon cooling, the PVA solution was combined with a solutioncomprising of divinylbenzene (DVB; 80%), 2,2′-azobisisobutyronitrile(AIBN), N-vinyl 2 pyrrolidione (N-VP) and/or N-vinylcaprolactam (V-CAP),methacryloxypropyltrimethoxysilane (MAPTMOS) and/orp-styryltrimethoxysilane (Styryl TMOS), and one or more of the followingcoporogens: 2-ethylhexanoic acid (2-EHA), toluene (HPLC grade),cyclohexanol (CXL), 1-methyl-2-pyrrolidinone (NMP). The resultingsolution was mixed using a mechanical stirrer with Teflon paddle under anitrogen flow or by continuous static mixing for approximately 20minutes or until the desired particle size, monitored periodically viamicroscopy and/or laser diffraction (Mastersizer 2000 MalvernInstruments, Westborough, Mass.), was obtained. The emulsification wasthen heated to 70-80° C. for 20 hours while stirring (mechanicalstirring with Teflon paddle). Upon cooling to 50° C., the suspension offormed particles was quenched with water and the particles were filteredand then washed consecutively with copious amounts of hot water (80°C.), RT water, and then methanol. The particles were then dried at 80°C. at a reduced pressure for 16 hours. Specific reagent amounts andreaction conditions are listed in FIG. 5. The specific surface areas(SSA), specific pore volumes (SPV) and the average pore diameters (APD)of these materials were measured using the multi-point N₂ sorptionmethod and are listed in FIG. 5 (Micromeritics ASAP 2420; MicromeriticsInstruments Inc., Norcross, Ga., or equivalent). The specific surfacearea was calculated using the BET method, the specific pore volume wasthe single point value determined for P/P₀>0.98, and the average porediameter was calculated from the desorption leg of the isotherm usingthe BJH method.

Example 2a

The particles of hybrid silica prepared according to Example 2 weresulfonated using the following process.

Hybrid particles were combined with concentrated sulfuric acid (10 mL/gparticles) and stirred at room temperature in a 1 L round-bottom flask.After stirring for 1 hour, the solution was slowly added to a stirredsolution of water (>100 mL/g particles), and the mixture was stirred for10 minutes. The modified hybrid silica particles were either filtered orsedimented overnight to remove residual small particles then filtered.The isolated particles were washed successively with water, methanol (J.T. Baker), and then dried at 80° C. under reduced pressure for 16 hours.Analytical data can be found in FIG. 6.

Example 2b

The particles of hybrid silica prepared from Example 2a were surfacemodified with mC18 silane using the following process.

Hybrid particles were fully dispersed in toluene (5-10 mL/g particles)then azeotropically stripped (111° C., 1 h) to remove residual waterfrom the material. Upon cooling <40° C., 1.5 micromoles of silane persquare meter of particle surface area and 1 equivalent (per mole silane)of imidazole were added to the particle/toluene mixture. The resultantmixture was refluxed for 4 hours. Once the reaction was cooled <40° C.,water (5 mL/g particles) was added to the flask and the mixture wasallowed to stir for 5 minutes. The modified hybrid silica particles werefiltered and washed successively with toluene, acetone, 1:1 v/vacetone/water, and acetone (5 mL/g particle), and then dried at 80° C.under reduced pressure for 16 hours. Analytical data can be found inTable 4. The loading of sulfonic acid groups was determined by titrationwith 0.1 N NaOH (Metrohm 905 Titrando autotitrator with 6.0280.300 pHelectrode; Metrohm, Hersau, Switzerland, or equivalent).

TABLE 4 mC18 Precursor Capacity Bonding ID Sample ID (meq/g) 3a 2a 0.823b 2b 0.82 3c 2c 0.81 3e 2e 0.85 3f 2f 0.81 3g 2g 1.04 3h 2h 1.01 3i 2i1.02

Example 3

A rat plasma (Equitech-Bio Inc., Kerrville Tex.) sample was spiked with10 analytes, to the concentrations given in Table 5. Analytes werepurchased from Analytes were purchased from Cerilliant® AnalyticalReference Standards, Round Rock, Tex., USA, a SIGMA-ALDRICH® Company.

A 1 mL sample was prepared using 500 μL of rat blood plasma(Bioreclamation IVT Westbury, N.Y. 11590), 460 μL of 4% H3PO4, andspiked with 40 μL of a solution of 10 analytes with the concentrationslisted in Table 5.

TABLE 5 Compunds Abbreviation Conc. (ng/mL) Tramadol TRA 100 TapentadolTAP 100 Benzoylecgonine BZE 100 7-Aminoclonazepam 7-AMI 20 PhencyclidinePCP 20 Fentanyl FEN 100 Flurazepam FLU 100 Methadone METH 20A-Hydroxyalprazolam AOHA 20 Diazepam DIAZ 100

The major class of phospholipids (PL) selected for monitoring arephosphatidylcholine (PC), making up approximately 70% of total humanplasma phospholipids. See Ismaiel, O. A. et al., “Investigation ofendogenous blood plasma phospholipids, cholesterol and glycerides thatcontribute to matrix effects in bioanalysis by liquidchromatography/mass spectrometry,” J. Chromatogr., B 2010, 878,3303-3316.

SPE protocols are shown in Table 6 below. The standard protocol (noconditioning or equilibration steps) for mixed mode solid phaseextraction (SPE) using Oasis MCX™ is shown on the left-hand side of thetable (MCX Protocol), whereas 4-step, 3-step and 2-step protocols inaccordance with the present disclosure are shown of the right-hand sideof the table.

TABLE 6 2-Step MCX Protocol 4-Step Protocol 3-Step Protocol ProtocolOasis MCX ^(™) SPE Matrix SPE Matrix SPE Matrix SPE Protocol RemovalRemoval Removal (bases with Protocol (bases Simplified Minimal pKa 2-10)with pKa 2-10) Protocol Protocol (bases with (bases with pKa 2-10) pKa2-10) Step 1-Load Step 1-Load Step 1-Load Step 1-Load Pre-treatedPre-treated Pre-treated Pre-treated sample sample sample sample (e.g.dilute (e.g. dilute (e.g. dilute (e.g. dilute plasma plasma 1:1 plasma1:1 plasma 1:4 1:1 with 4% with 4% with 4% with 2% H₃P0₄(aq)) H₃P0₄(aq))H₃P0₄(aq)) H₃P0₄ and 2% Formic Acid in Methanol) Step 2-Wash 1 Step2-Wash 1 Step 2-Wash 2% Formic Acid 2% Formic Acid 2% Formic Acid (aq)(aq) in Methanol Step 3-Wash 2 Step 3-Wash 2 100% Methanol 100% MethanolStep 4-Elute Step 4-Elute Step 3-Elute Step 2-Elute 5% NH₄OH in 5% NH₄OH5% NH₄OH 5% NH₄OH Methanol in 75/25 (v/v) in 75/25 (v/v) in 75/25 (v/v)Acetonitrile/ Acetonitrile/ Acetonitrile/ Methanol Methanol Methanol

Typical loading volume is 25-750 μL (200 μL preferred) for WatersμElution plate (Waters Corporation, Milford, Mass., USA) (2 mg sorbentmass format). Typical washing volume is 200-400 μL. Typical elutionvolume is 25-100 μL (50 μL preferred). Typical dilution volume, ifdesired, is 50-150 μL. In general, volumes are scaled with sorbent massor volume.

Solutions used for the protocols are as follows:

-   -   2% formic acid (aqueous) is a solution 98/2 (v/v) water/formic        acid    -   2% formic acid in methanol is a solution 98/2 (v/v)        methanol/formic acid    -   5% NH₄OH in methanol is a solution 95/5 (v/v) methanol/NH₄OH        from concentrated NH₄OH    -   5% NH₄OH in 75/25 acetonitrile/methanol is a solution        71.25/23.75/5 (v/v/v) acetonitrile/methanol/NH₄OH from        concentrated NH₄OH    -   4% H₃PO₄ (aq) is an aqueous solution of 96/4 (v/v) water/H₃PO₄        from concentrated H₃PO₄.    -   2% H₃PO₄ and 2% formic acid in methanol is a solution of 96/2/2        (v/v) methanol/H₃PO₄ from concentrated H₃PO₄/formic acid.

The protocol for analyte capture and matrix removal is as follows:samples are pre-treated using 4% H₃PO₄ (aq) in a 1:1 ratio or using 2%H₃PO₄ and 2% formic acid in methanol in a 1:4 ratio, and then loadedinto a plate well or cartridge device. For MCX and 4-Step Protocols thefirst wash is an acidic aqueous solution and the second wash is a high %organic solution. For the 3-Step Protocol, the contents of the twowashes of the 4-Step Protocol have been combined into an acidifiedorganic/aqueous solution. The wells/cartridges are ‘pulled dry’ prior tothe elution step. The final elute step is a basified high organicsolution with a protic solvent and a polar aprotic solvent, typicallywith more than 60% acetonitrile, which is provided to removephospholipids. The protic solvent content, typically around 10% to 30%methanol, is used to achieve acceptable analyte recovery.

Phospholipid and Analyte Analyses: 2 μL of the pre-treated sample wasinjected on an ACQUITY CSH C18 1.7 μm 2.1×100 mm UPLC column (WatersCorporation) and analyzed with a XEVO TQ-S Mass Spectrometer (WatersCorporation). UPLC mobile phases for lipids quantification and analyterecovery tests (gradient conditions) are shown in Tables 7 and 8, wheremobile phase A is 0.1% formic acid in water and mobile phase B is 0.1%formic acid in acetonitrile.

TABLE 7 Time (min) Flow Rate (mL/min % A % B Curve Initial 0.5 90 10Initial 1 0.5 90 10 6 4 0.5 30 70 6 4.1 0.5 5 95 6 7 0.5 5 95 6 7.5 0.590 10 6 8 0.5 90 10 6

TABLE 8 Time (min) Flow Rate (mL/min % A % B Curve Initial 0.6 95 5Initial 5 0.6 10 90 6 5.1 0.6 95 5 6 6 0.6 95 5 6

Conditions for the Xevo TQ-S Mass Spectrometer are as follows:

-   -   Capillary voltage: 3.0 kV    -   Cone voltage: 50 V    -   Desolvation temperature: 500° C.    -   Desolvation gas flow: 100 L/hr    -   Cone gas flow: 150 L/hr    -   Nebulizer: 7.0 Bar    -   Collision gas flow: 0.25 ml/min

The results are as follows. Using the MCX Protocol, the particle ofExample 1 performs comparably for phospholipid removal to Oasis MCX™.However, using the 4-step Protocol, the Coated Oasis MCX™ particleachieved 64% additional phospholipid removal vs. Oasis MCX™ The 4-stepProtocol used on the Coated Oasis MCX™ achieved an additional 81%phospholipid removal vs. Oasis MCX™ using the MCX Protocol. FIG. 1 showstotal phospholipid content (TIC peak area) for Oasis MCX™ compared tothe particle of Example 1. Both sorbents were tested with the MCXProtocol (left) and the 4-step Protocol (right) (90/10acetonitrile/methanol used in the elution step).

With regard to analyte recovery (pre- and post-spike of analytestandards) data shown in FIG. 2 for both materials tested (i.e., OasisMCX™ and particle of Example 1) under the MCX Protocol demonstratessimilar loading/ion exchange capacity for the materials.

Using the Oasis MCX™ Protocol, particle of Example 2 performs comparablyfor phospholipid removal to Oasis MCX™. Using the 4-step Protocol (5%NH₄OH in 90/10 ACN/MeOH final elution), however, the particle of Example2 achieved 69% additional phospholipid removal vs. Oasis MCX™ using MCXProtocol. Moreover, as seen in FIG. 3, for the 3-Step Protocol, theparticle of Example 2 achieved an additional 84% phospholipid removalvs. Oasis MCX™ using the MCX Protocol. In particular, FIG. 3 shows totalphospholipid content (TIC peak area) for Oasis MCX™ compared to iparticle of Example 2, with both sorbents being tested with the MCXProtocol (left) and the 3-step Protocol (right). The 3-Step Protocol isvery effective with the particle of Example 2 for removing phospholipidsfrom the final elute fraction.

Furthermore, as seen from FIG. 4, analyte recovery (pre- and post-spikeof analyte standards) is comparable for the Oasis MCX™ and the particleof Example 2 under the MCX Protocol and the 3—Step Protocol, indicatingthat both materials perform similarly under each Protocol and ionexchange capacity for the particle of Example 2 is comparable to theOasis MCX™ material.

1-34. (canceled)
 35. Inorganic/organic hybrid sorbent particlescomprising an organic copolymer that comprises at least one hydrophilicorganic monomer, at least one hydrophobic organic monomer, and at leastone alkenyl-functionalized organosilane monomer, wherein the particlesare modified by the addition of a C4-C60 alkyl component.
 36. Theinorganic/organic hybrid sorbent particles of claim 35, wherein theparticles are modified by hydrolytic condensation of aC4-C60-alkyl-functionalized organosilane compound.
 37. Theinorganic/organic hybrid sorbent particles of claim 36, wherein theC4-C60-alkyl-functionalized organosilane compound is a compound of theformula RSiZ₁Z₂Z₃, where R is C4-C60 alkyl and where Z₁, Z₂ and Z₃ areindependently selected from Cl, Br, I, C1-C4 alkoxy, C1-C4 alkylamino,and C1-C4 alkyl, although at most two of Z₁, Z₂ and Z₃ can be C1-C4alkyl.
 38. inorganic/organic hybrid sorbent particles of claim 36,wherein the C4-C60-alkylorganosilane is selected fromoctadecyltrimethoxysilane, octadecyltriethoxysilane,octadecyltrichlorosilane, octadecyltri(dimethylamino)silane,methyloctadecyldimethoxysilane, methyloctadecyldiethoxysilane,methyloctadecyldichlorosilane, methyloctadecyldi(dimethylamino)silane,dimethyloctadecylmethoxysilane, dimethyloctadecylethoxysilane,dimethyloctadecylchlorosilane, dimethyloctadecyldimethylaminosilane,diisopropyloctadecylmethoxysilane, diisopropyloctadecylethoxysilane,diisopropyloctadecylchlorosilane,diisopropyloctadecyldimethylaminosilane, octyltrimethoxysilane,octyltriethoxysilane, octyltrichlorosilane,octyltri(dimethylamino)silane, methyloctyldimethoxysilane,methyloctyldiethoxysilane, methyloctyldichlorosilane,methyloctyldi(dimethylamino)silane, dimethyloctylmethoxysilane,dimethyloctylethoxysilane, dimethyloctylchlorosilane,dimethyloctyldimethylaminosilane, diisopropyloctylmethoxysilane,diisopropyloctylethoxysilane, diisopropyloctylchlorosilane,diisopropyloctyldimethylaminosilane, butyltrimethoxysilane,butyltriethoxysilane, butyltrichlorosilane,butyltri(dimethylamino)silane, methylbutyldimethoxysilane,methylbutyldiethoxysilane, methylbutyldichlorosilane,methylbutyldi(dimethylamino)silane, dimethylbutylmethoxysilane,dimethylbutylethoxysilane, dimethylbutylchlorosilane,dimethylbutyldimethylaminosilane, diisopropylbutylmethoxysilane,diisopropylbutylethoxysilane, diisopropylbutylchlorosilane, anddiisopropylbutyldimethylaminosilane.
 39. The inorganic/organic hybridsorbent particles of claim 35, wherein the alkenyl-functionalizedorganosilane monomer is selected from 3-(trimethoxysilyl)propylmethacrylate (MAPTMOS) and vinyltriethoxysilane (VTES).
 40. Theinorganic/organic hybrid sorbent particles of claim 35, wherein thehydrophilic organic monomer comprises a monomer having the followingformula,

where n ranges from 1-3.
 41. The inorganic/organic hybrid sorbentparticles of claim 35, wherein the hydrophobic organic monomer comprisesdivinylbenzene and, optionally, styrene.
 42. The inorganic/organichybrid sorbent particles of claim 35, wherein the organic copolymerfurther comprises an organic monomer that comprises one or moresulfonate groups.
 43. The inorganic/organic hybrid sorbent particles ofclaim 35, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more sulfonate groups. 44.The inorganic/organic hybrid sorbent particles of claim 35, wherein theorganic copolymer further comprises an organic monomer that comprisesone or more quaternary ammonium groups.
 45. The inorganic/organic hybridsorbent particles of claim 35, wherein the organic copolymer furthercomprises an organic monomer that comprises one or more —R₁N⁺R₂R₃R₄groups, where R₁ is a C1-C8 alkylene group, and R₂, R₃ and R₄ are C1-C8alkyl groups and may be the same or different.
 46. The inorganic/organichybrid sorbent particles of claim 35, wherein the organic copolymerfurther comprises a divinylbenzene monomer that comprises one ormore-R₁N⁺R₂R₃R₄ groups, where R₁ is a C1-C8 alkylene group, and R₂, R₃and R₄ are C1-C8 alkyl groups and may be the same or different.
 47. Theinorganic/organic hybrid sorbent particles of claim 35, wherein theorganic copolymer further comprises an organic monomer that comprisesone or more carboxyl groups.
 48. The inorganic/organic hybrid sorbentparticles of claim 35, wherein the organic copolymer further comprises adivinylbenzene monomer that comprises one or more carboxyl groups. 49.The inorganic/organic hybrid sorbent particles of claim 35, wherein theorganic copolymer further comprises an organic monomer that comprisesone or more primary, secondary or tertiary amine groups.
 50. Theinorganic/organic hybrid sorbent particles of claim 35, wherein theorganic copolymer further comprises a divinylbenzene monomer thatcomprises one or more piperazine groups.